Antipsychotic Prochlorperazine Restrains Bladder Cancer Growth by Regulating cell proliferation and SRC-MEK-ERK Pathway

doi:10.21203/rs.3.rs-3420728/v1

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Antipsychotic Prochlorperazine Restrains Bladder Cancer Growth by Regulating cell proliferation and SRC-MEK-ERK Pathway

https://doi.org/10.21203/rs.3.rs-3420728/v1

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The high incidence of bladder cancer and inconvenient life quality prompt us to find new therapeutic candidates. Prochlorperazine, mainly as an effective sedative, antiemetic reagent, was shown to exhibit anti-cancer activity in several studies, putting it up as a therapeutic candidate for bladder cancer. Network pharmacologic approaches is a high valuable tool in predicting rational drug targets within disease signaling module. Network based protein-protein interactome prediction, GO annotation and KEGG pathway enrichment analysis showed that prochlorperazine might affect bladder cancer growth through multiple signaling pathways. Cellular function experiments revealed that prochorperazine inhibited cell proliferation in several bladder cancer cell lines and in vivo mouse xenograft test confirmed its significant inhibition effect on BC. Western blotting analysis demonstrated that prochlorperazine treatment markedly modulated the expression and phosphorylation levels of MAPK1（ERK2）、MAP2K1（MEK1）and SRC, showing the possible molecular mechanism via the SRC-MEK-ERK pathway in BC cancer. These studies indicated the potential inhibitory impact of prochlorperazine and provided new ideas for the pathogenesis and treatments of BC.

network pharmacology

prochlorperazine

bladder cancer

therapeutic candidate

Bladder cancer (BC) is one of the most prevalent cancer overworld, especially in developed countries. The incidence of BC is particularly high in men, which is 3-4times more than in women. Most of BCs are non-muscle-invasive bladder cancers (NMIBC), which have better favorable prognosis than the more malignant muscle-invasive bladder cancers (MIBC). The first-line treatment of NMIBCs is transurethral resection, usually followed by intravesical delivery of chemotherapeutics such as mitomycin C or bacillus Calmette-Guerin (BCG) into the bladder lumen. As for the muscle-invasive BCs, radical cystectomy followed by a period of intake of cisplatin-based neoadjuvant are the common regimens. The 5-year survival rate is about 50% for patients who underwent cystectomy. However, these patients often suffered a lot from the inconvenience of life and so that many patients disfavored cystectomy.

Despite that platin-based chemotherapy and immune checkpoint inhibitors have improved the quality and survival rate of BC patients, the mortality rate is still high mainly due to metastasis. It is therefore urgent and essential to find new therapeutic candidates for bladder cancer patients.

Prochlorperazine (PCZ)is a phenothiazine derivative that is commonly used as a typical antipsychotic in treating schizophrenia and other psychotic disorders[1]. It can bind to but do not activate dopamine receptors and thereby blocking the actions of dopamine or exogenous agonists. It is usually used to treat cancerous patients who is suffering nausea, vomiting or headaches because of chemotherapies such as cisplatin (CDDP) treatment[2].

As earlier as in 1984, Sheila Mac Nail and the colleagues reported that PCZ could inhibit DNA synthesis and therefore inhibit B16 melanoma cell proliferation[3]. Several other studies also indicated that it could alleviate cancer growth in such as melanoma[4], glioblastoma[5], acute leukemia[6] and non-small cell lung carcinoma[7] et al. PCZ showed good binding affinity upon KRAS-mutant proteins and, along with radiation, PCZ treatment caused DNA damage-induced apoptosis in KRAS-mutant NSCLC, proposing it as a novel radiosensitizer in NSCLC[7]. The potential role of PCZ in bladder cancer has not been explored yet.

Network pharmacology is a highly powerful and effective tool in predicting drug targets and establishing the pragmatic network models based on public databases[8]. It helps to construct complex ‘drug-target-disease’ network via high-throughput screening and bioinformation for drug development in cancer and other diseases. In order to explore the capability of prochlorperazine on inhibiting bladder cancer growth as a new therapeutic, in this study, we began with network pharmacology analysis to see the potential capacity and mechanisms of prochlorperazine in bladder cancer, and then validated the impact on bladder cancer cell viability in vitro and further in vivo.

2.1 Bioinformatic Analysis of the Relationship between PCZ and Bladder Cancer

The targets of prochlorperazine were retrieved from the databases including Drugbank（https://go.drugbank.com）、BATMAN-TCM（http://bionet.ncpsb.org.cn/batman-tcm）、SwissTargetPrediction（http://swisstargetprediction.ch）and STITCH（http://stitch.embl.de）. Meanwhile, the targets of bladder cancer-related molecules were obtained from the two databases named GeneCards（https://www.genecards.org）and DisGeNET（https://www.disgenet.or). The common targets between prochlorperazine and bladder cancer were screened out using the online tool Venny 2.1 (http://bioinfogp.cnb. csic.es/tools/venny/index.html).

Then the common targets obtained were imported into the STRING database (https://www.string-db.org/) to survey their interaction relationship under the defined conditions of organism “Homo sapiens” and high confidence (interaction score > 0.95). After exported, these resulting data was then imported into Cytoscape 3.9.0 to merge and calculate the intersection of PPI network and core genes...

For functional annotation of gene ontology (GO) and analysis of KEGG pathways of these core genes, we performed enrichment analysis using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt, http://www.webgestalt.org/option.php).

And in order to understand the complex associations among prochlorperazine, the potential targets and bladder cancer, we constructed and analyzed the relationship networks using Cytoscape (https://www.nigms.nih.gov/). It provides a powerful set of data integration, analysis, and visualization functions to analyze the complicated networks.

To understand the possible binding properties between PCZ and the selected targets, molecular docking simulation was analyzed in Autodock vina 1.1.2. This program was used for network pharmacology-based prediction and analysis. It could also illustrate the mechanism of a ligand acting on a complex molecular network by applying high-precision docking simulation and molecular pathway map.

2.2 Cell Culture

Human bladder Cancer cell lines HT1376, T24, EJ and the mouse BC cell line MB49 were purchased from Shanghai FUHENG Biotechnology limited company and cultured in the special media for HT1736 (Procella, Wuhan, China) or PMRI1640 medium (Gibco, invitrogen, USA) supplemented with 10%FBS (SeraPro, Germany) for the other cell lines.

2.3 Drugs and Reagents

Prochlorperazine was purchased from Jiangsu Aikon Biomedical R&D Co., Ltd, Jiangsu, China. CCK8 assay kit was purchased from Beyotime biotech Co. Ltd, Shanghai, China. DCFH-DA was purchased from SigmaAldrich, USA.

Antibodies for MEK1/2/p-MEK1/2, ERK1/2/p-ERK1/2 and Src/p-Src were all purchased from Chengdu Zen-Bioscience Co., Ltd. (“Zen-Bioscience”), Sichuan, China.

2.4 Cell Viability Analysis

Cell viability of BC cells was measured by CCK8 assay kit according to the instruction (Beyotime, Shanghai, China). Cells were plated onto 96well plates in 6 technical replicates at a density of 5000cells/well the day before and treated with 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50µM of Prochlorperazine in media for 24-96hrs. Afterward, cells were incubated with CCK8 regent for another 2hrs and then the absorbance at 450nM was measured. IC50 was calculated on GraphPad (La Jolla, USA) Prism9.0 using non-linear curve fitting model. Viability of control was set to 100% and the viability of treated cells was normalized to control. The assay was repeated for three times.

2.5 Cell Migration Assay

Cell migration assay was conducted with Transwell insert. 5X10⁴cells in FBS free medium were plated on the upper chambers of the transwell inserts and FBS containing medium supplemented with different concentrations of prochlorperazine were added in the lower chambers. Cells migrated onto the outer side of the membrane were fixed in 4% PFA and stained with 0.1% crystal violet. Photos were taken and the number of cells on the filter was counted.

2.6 EdU Staining Assay

EdU staining was done with a staining kit (Beyotime, Shanghai, China) as the instructions. Briefly, Cells were plated into 24well plates in replicate at a density of 2000cells/well the day before treatment and intervened with 0, 2.5, 5, 10µM of Prochlorperazine in media for 48hrs. And then cells were incubated with 10µM 5-ethynyl-2_-deoxyuridine (EdU) working solution for another 2hrs, fixed in 4%PFA, permeablized with 0.3% Trixton-X100, followed by Click reaction with Alexa555-azide under CuSO4-catalyzed condition to visualize the EdU-labeled nuclei.

2.7 ROS Staining Assay

For cellular ROS level detection, cells treated with different concentrations of prochlorperazine were stained with 10µM DCFH-DA (2’, 7’-dichlorofluorescein diacetate) in PBS for 30 min in dark at 37°C, washed and examined under an Olympus microscope equipped with a fluorescence detection module.

2.8 Western Blotting Analysis

After treated with different concentrations of prochlorperazine, cells were lysed by RIPA lysis buffer to extract the cellular proteins and were electrophoresis separated in 12% SDS-PAGE gel and then transferred onto the 0.22µm PVDF (polyvinylidene difluoride) membrane. Primary antibodies were incubated with the membrane overnight at 4°C following blocking with 5% BSA, washed and then hybridized with the HRP (Horseradish peroxidase)-conjugated secondary antibodies, visualized with ECL substrate (Tanon, Shanghai, China). The GAPDH antibody was used as the internal reference control.

2.9 In Vivo Animal Study

Animal studies were conducted following the National Academy of Sciences rules for the care and use of laboratory animals, approved by the Institutional Animal Care and Use Committee (IACUC) of Nanchang Royo Biotech Co., Ltd. (RYE:2022040301). Female BALB/c athymic nude mice of 4-6 weeks old were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co. LTD.

To establish a tumor xenograft model, 5 × 10⁶-1×10⁷cells diluted in 200μl PBS were subcutaneously injected into the anterior scapular region of each mouse. Tumor sizes were measured with a caliper twice a week, and the tumor volumes were calculated as follows: tumor volume (mm³) = 0.5 × length × width². When the tumor volumes reached about 50-100mm³ about 7 days later, the nude mice were randomized with each group containing 5 mice: control, prochlorperazine group (2mg/kg, I.P., daily) and the cisplatin positive control group (5mg/kg, I.P.,once a week). Tumor weights were weighed out using an analytical balance at the end of the experiment after 21 days of treatment.

2.10 Statistics

Data were expressed as mean ± SD from at least three independent experiments. Comparisons between groups were analyzed using the Student’s t-test and one-way analysis of variance (ANOVA) by GraphPad Prism 9.0 (GraphPad Software Inc., California, USA). P < 0.05 was considered to indicate a statistically significant difference.

3.1 Identification and Enrichment Analysis of Common Targets of Prochlorperazine and Bladder cancer

Structural information of Prochlorperazine was obtained from the NCBI PubChem website(https://pubchem.ncbi.nlm.nih.gov)(FIG1A). We retrieved a total of 250 prochlorperazine-related targets from the Drugbank, BATMAN-TCM, SwissTargetPrediction and STITCH databases after wiping off the duplicates. And 4381 bladder cancer-related target genes were obtained from DisGeNET and GeneCards databases. After being inputted into Venny 2.1, we found that there were 123 common genes between prochlorperazine-related and bladder cancer-related targets after eliminating the redundancy (FIG1B).

To obtain the interaction relationship, the 123 common targets were then imported into the STRING database. Scoring value > 0.95 was selected as the high confidence basis for protein interactions. The interaction network has 123 nodes and 75 edges (Fig. 2A). The obtained data from STRING TSV data were imported into Cytoscape, and the results showed a total of 25 nodes and 39 edges. Then, the most related 10 targets including SRC、ESR1, ARRB1, MAPK1, PRKCD, FYN, ESR2, MAP2K1, PTK2B, KCNQ1 were further screened as the center genes, most of which were involved in the MAPK signaling pathway (Fig. 2B).

3.2 Enrichment Analysis of the Common Target

In order to obtain a more detailed understanding of the potential mechanisms of PCZ acting on bladder cancer, GO function and Pathway analysis were performed using WebGestalt(http://www.webgestalt.org/option.php). According to the annotation results, the top five affected biological processes were biological regulation, response to stimulus, multicellular organismal process, metabolic process and cell communication (Fig. 3A). And the main molecular function involved was protein binding. As for pathway analysis, the common targets took part in 10 KEGG pathways with significant P-value < 0.01 including pathways in cancer, Rap1 signaling pathway, neuroactive ligand-receptor interaction and so on.

3.3 Interaction Prediction

In order to understand the complex relationships among compound, targets, and diseases, we used Cytoscape to construct and analyze the networks. As shown in FIG4, this compound (prochlorperazine), disease (bladder cancer), targets and pathways interaction network have 135 nodes and 405 edges. The nodes labeled with red, blue, orange and green respectively represent prochlorperazine, bladder cancer, related pathways and targets, the lines represent the interactions among them.

To further elucidate the potential interactions, MAPK1 and MAP2K1 as the target gene were mapped to the compound for molecular docking. The affinity value was specified as the docking score. An affinity less than − 7kcal/mol indicates a strong binding activity, that is the lower value means the stronger binding. The binding energies of prochlorperazine to MAPK1 and MAP2K1 were − 7.4kcal/mol and − 6.9kcal/mol respectively. The combination models of prochlorperazine and these two genes were shown in FIG.5 in 3D graphs. The results indicated that prochlorperazine had good binding activities to MAPK1 and MAP2K1.

3.4 Regulation of Prochlorperazine on Bladder Cancer Cell Viability

To evaluate the actual effects of prochlorperazine on bladder cancer cell growth, Cell viability was measured using CCK8 assay with human bladder cancer cell lines such as HT1376, T24, EJ and the MB49 mouse BC cell line. Prochlorperazine was applied to cells at different concentrations for 24, 48 and 72 hours. It showed that prochlorperazine exerted great cytotoxicity on the BC cell lines with the Half-maximal inhibitory concentrations (IC50s) ranged from 22.38 to 45.99µM at 24hrs duration, 15.97–30.91µM at 48hrs duration and 11.5-30.78µM at 72hrs duration. Among these cell lines, HT1376 seemed to be the most sensitive to prochlorperazine intervention (FIG6A.B).

EdU incorporation assay results in HT1376 confirmed that PCZ supplementation in the growth media alleviated the DNA incorporation process, verifying the inhibitory effect of PCZ on bladder cancer cell proliferation (FIG6C.D).

3.5 Influence of Prochlorperazine on Bladder Cancer Cell Apoptosis

In the view of the above data, we aimed to disclose whether cell apoptosis or senescence process were also affected by PCZ for its cytotoxic function. As oxidative stress could induce mitochondrial damage and lead to cell death, cellular reactive oxygen species levels (ROS) in HT1376 were examined. As indicated by DCFH-DA staining, PCZ treatment increased cellular ROS levels markedly (FIG7A.B).

3.6 Modulation of Prochlorperazine on the Potential Target Genes

The expression and phosphorylation levels of MAPK1(ERK2) and MAP2K1(MEK1) in bladder cancer cells were further examined to clarify the possible molecular mechanisms of prochlorperazine. Western blotting analysis demonstrated that it significantly down-regulated the expression and phosphorylation levels of MAPK1(ERK2) while compensatorially improved the expression and phosphorylation levels of MAP2K1(MEK1) after 48hrs incubation of prochlorperazine compared to the non-treated control group, further verifying the predictive results of network pharmacology (Fig. 8). These results indicated that PCZ might possibly make an impact on bladder cancer growth via affecting the activities of MAPK1 or MAP2K1.

3.7 Efficacy of Prochlorperazine on Bladder Cancer Progression in Mice Xenograft

As shown in the tumor growth curves and the T/C percent values which indicating the relative tumor growth rate, the xenografts grew significantly slower in 2mg/ml PCZ treated MB49 syngeneic tumor bearing mice. Significant reduction of tumor weights was observed at end, compared to the untreated controls (Fig. 9). No obvious change in mice body weight was observed, suggesting no systemic toxic response with PCZ.

Bladder cancer has various histological types including urothelial carcinoma. Surgery is the main treatment, and chemotherapy is often used before and after resection. Although there are various regimens for bladder cancer, both early surgical treatment and subsequent combined chemotherapy and radiotherapy are extremely harmful to patients. Prognosis for BC is often not ideal.

Prochlorperazine, as an antiemetics and antipsychotics, is generally used as an adjuvant in treating the side effects of chemotherapy. It has been proposed to temporary inhibit endocytosis for numerous diseases including epilepsy, viral or bacterial infection and cancer by altering receptor distribution in vivo[9, 10], supposing a potential role for PCZ as a non-selective dynamic and reversible inhibitor in clinic.

In this article, we for the first time explored the probability of prochlorperazine on inhibiting bladder cancer based on network pharmacology analysis with several public databases and probed the possible signaling pathways and networks. Cellular viability detection using CCK8 regent and EdU incorporation assay and as well as the in vivo mouse xenograft models all demonstrated its inhibitory effect in BC cancer growth. We also found that PCZ could dramatically enhance the ROS levels of BC cell, indicating its potential ability on cell apoptosis. Western blotting analysis further illuminated the possible inhibitory effect of PCZ on MAPK signaling pathway, suggesting a probable molecular mechanism of PCZ in suppressing BC cancer.

The RAF-MEK-ERK MAPK cascade is the most intensively studied signaling pathway that is key for a variety of cellular functions such as cell proliferation and survival. High MAPK activity was generally restricted to tumor cells adjacent to the invasive edge[11], contributing to the invasion, tumor stemness and EMT process of BC[12–15]. MAPK1 (also named ERK2) and MAP2K1 (also named MEK1) are two core components of this well-known MAPK signaling pathway and are closely related to the occurrence and development of BC. Significantly higher MAPK activity was found in muscle-invasive bladder cancer (MIBC) than in non-muscle-invasive bladder cancer (NMIBC). MAPK1(also named ERK2) is a prototypical mitogen-activated protein kinase (MAPK) family member and phosphorylated ERK is an indicator of MAPK pathway activation. Molecular docking simulation has shown that MAPK1(ERK2) and MAP2K1(also named MEK1) have high affinity with prochlorperazine and western blotting analysis further demonstrated that prochlorperazine (PCZ) could reduce the expression and phosphorylation of MAPK1(ERK2), proposing the possibility of PCZ to affect BC cancer via binding to MAPK1(ERK2). Our western blotting analysis also detected the increasing levels of MEK/p-MEK and SRC/p-SRC (less significant than ERK/p-ERK) by PCZ treatment. The reason for this opposite impact might due to a compensatory effect. PCZ was previously shown to exert anti-proliferative effect in murine cancer model via inhibiting AKT activation[6], and could sensitize mice xenografts to radiotherapy also via downregulation of Ras/Raf/MEK/ERK pathway[7]. Overexpression or activation of Src tyrosine kinase, a non-receptor tyrosine kinase that reside upstream of MAPK pathway, was shown to promote tumor development and progression including bladder cancer, breast cancer and lung cancer et al[16, 17]. These findings together certified the possibility of PCZ in inhibiting BC cancer progression by regulating the MAPK signaling pathway.

The capability of PCZ to restrain BC cancer growth and exerting no obvious toxicity might provide helpful clues on developing novel and effective therapies for BC. Although as a widely accepted antipsychotics and an adjuvant in cancer patient, our preliminary study in this article rationalizes the repurposing of prochlorperazine to treat bladder cancer as a new therapeutic in clinical. Repurposing of clinically practical drugs could bypass or shorten the development process for their already learned pharmacological and toxicological studies and so that accelerate the translational use of the drug in different diseases.

There are limitations for our research in that just a few in vitro cellular growth assays using cell lines have been carried out and the xenograft model was established with cell lines, which lacked tumor heterogeneity and human tumor environment. In vivo studies with patient-derived tumor xenograft models, which poses high fidelity consistent with the patient pathology, would be a further adaptive choice to elucidate the actual effect of PCZ on bladder cancer. Besides, the molecular mechanism probing of this study was relatively simple. Based on the network pharmacologic PPI interaction analysis and KEGG pathway annotation of the common targets of both prochlorperazine and bladder cancer, it is conjectable that prochlorperazine might exert multiple effects on bladder cancer growth by regulating several pathways. The possible molecular mechanism of prochlorperazine in inhibiting tumorigenesis also could be related to the Rap1 signaling pathway as indicated in KEGG pathway enrichment result. Evidences showed that Rap1 signaling pathway is one of the most frequently activated pathways in cancer[18, 19] and is involved in cell-cell junction, cell differentiation and tumor angiogenesis in several cancer types[20–22]. More extensive analysis on signaling pathways are needed to be explicit to support the findings and widen the application of prochlorperazine.

Ethics approval and consent to participate：The ethical consent of the Second Affiliated Hospital of Nanchang University and Nanchang ROYO Biotech company were obtained for this article. And written informed consent from all authors also were obtained.

Consent for publication：All authors agree to publish.

Competing interests: The authors declare no competing interests.

Funding: This study was supported by the National Natural Science of China (No. 82060465 to X.B.L.), the Natural Foundation of Jiangxi Province (NO. 20212ACB206023 to X. B.L. and No. 20192BCD40003 to Y. Q. H), and the Science Foundation of Jiangxi Provincial Education Department (No. GJJ150231 to Y. Q. H.).

Acknowledgements: All authors contributed to either data analysis, drafting or revising of the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Availability of data and material：The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Antipsychotic Prochlorperazine Restrains Bladder Cancer Growth by Regulating cell proliferation and SRC-MEK-ERK Pathway

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Abstract

Figures

1 Introductions

2 Materials and Methods

3 Results

3.1 Identification and Enrichment Analysis of Common Targets of Prochlorperazine and Bladder cancer

3.2 Enrichment Analysis of the Common Target

3.3 Interaction Prediction

3.4 Regulation of Prochlorperazine on Bladder Cancer Cell Viability

3.5 Influence of Prochlorperazine on Bladder Cancer Cell Apoptosis

3.6 Modulation of Prochlorperazine on the Potential Target Genes

3.7 Efficacy of Prochlorperazine on Bladder Cancer Progression in Mice Xenograft

4 Discussion

Declarations

References

Additional Declarations

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