Screening of a FDA-Approved Compound Library Identifies Menadione in Regulating Colon Cancer Programmed Death via MAPK8 Cascades

Screening of a FDA-Approved Compound Library Identifies Menadione in Regulating Colon Cancer Programmed Death via MAPK8 Cascades | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Screening of a FDA-Approved Compound Library Identifies Menadione in Regulating Colon Cancer Programmed Death via MAPK8 Cascades Weiwei Song, Liyuan Cao, Lei Ding, Xueqi Ma, Pengfei Gu, Wenbo Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1406834/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Colorectal cancer (CRC) is a conventional gastrointestinal malignant tumor, ranking third among all type of tumors and the fifth in clinical mortality. Programed cell death including apoptosis, autophagy and necroptosis that can be distinguished by their morphological and physiological differences. Although various researches and strategies were developed for anti-cancer drugs, there still lack of the effective therapies trigger cell programmed death to eliminate malignant colon tumor cells. In this study, we explored novel agents for inducing the programmed cell death through drug repurposing. We generated a high-throughput screening of a FDA Approved Drug Library, and identified Menadione, a synthetic analogue of vitamin K, as a promising candidate in regulating colon tumor programmed death. To further investigate the underlying mechanisms of Menadione in tumor programmed death, the selected gene of PCR array were performed and the data analysis showed significantly up-regulated gene by Menadione were ATG7 and MAPK8. The Go enrichment analysis also certify that MAPK8 may be an important regulation gene for Menadione induced cell apoptosis and necrosis in colorectal cancer. MAPK8 participated in multiple signaling pathways in GO enrichment analysis and KEGG pathway enrichment analysis, further demonstrated the key regulatory role of MAPK8. Thus, we identified Menadione may be a potential compound for MAPK8-targeting cascade. Owing to their FDA-approved status, Menadione might be used rapidly in the clinical treatment of tumor therapy. We demonstrated and provided new insights into the anti-cancer drug strategies. Colorectal cancer Programmed cell death Drug repurposing Menadione MAPK8 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women worldwide [ 1 ] Globally, 1.36 million people are affected accounting for nearly 10% of cancer prevalence [ 2 ] . It remains the second leading cause of cancer worldwide [ 3 ] . The most widely available treatments for managing colon cancer include surgery, radiation therapy, and chemotherapy. [ 4 ] Although some progress has been made in various of researches and development of CRC drugs in recent years, there are still many limitations and challenges, such as the side effects of chemotherapy and radiation, the limitation of targeted therapy because of KRAS or BRAF gene mutations, and the drug resistance of tumor cells caused by long-term drug medication. [ 5 ] In spite of their conceptual promise, more than one-third of colon cancer patients are often incurably. [ 6 ] With the development of the science, increasing attentions have been paid to the treatment of cancer by inducing the programmed death of tumor cells to regulate tumorigenesis. [ 7 , 8 ] The implementation of new therapeutic options for the management of colon cancer mandates a revisit to programmed cell death with an emphasis on the mechanisms. [ 9 ] TNF-α is known to act as a necroptosis inducer. [ 10 ] However, there are many problems in the application of TNF-α in clinical tumor treatment. For example, multiple organ failure caused by possible inflammatory cytokine storms is a major problem.. [ 11 ] Thus, developing the novel compounds to induce cell death, exploring the signaling pathways according to molecular basis of colon carcinogenesis and developing new drugs targeting them are necessary. Given the high attrition rates, a huge sum of costs and long cycle of discovery and development of new drugs, the use of "old" drugs to treat common and rare diseases is gradually becoming an attractive proposition. [ 12 ] Repurposing well-studied preclinical, clinical, and approved compounds holds the greatest potential to swiftly move a drug candidate from the bench to the clinic. [ 13 ] An FDA-approved compound library (TargetMol L4200) was well-studied compounds, many of which already possess extensive human safety data. [ 14 ] For instance, based on FDA drug library, thalidomide has been found to be used in the treatment of systemic lupus erythematosus and multiple myeloma. [ 15 ] Meanwhile, new drug strategies also could be focused on the regulation of cell death pathways, contributing to a better comprehension of cancer pathogenesis and therapeutics. [ 16 ] Supportive evidence has increasingly revealed the prime molecular mechanisms of each cell death subroutine and thus providing a succession of possible targets in cancer therapy, w hereas the complicated relationships between different cell death subroutines still remain to be elucidated. [ 17 ] Summary of above mentioned, drug repurposing through screening a FDA-approved drug library via programmed cell death would be the feasible method to develop novel drug for colorectal cancer. MAPK8(Mitogen-activated JNK1) is a mumber of MAPK family and JNK family encoding by a MAPK8 gene. MAPK family is one of serine-threonine protein kinases family that plays an important role in physiological and pathological process. [ 18 ] MAPK8 as a member of the MAPK family can activate and phosphorylate transcription factors (such as AP-1), thereby activating downstream genes that play an important regulatory role in cell proliferation, differentiation, death, and inflammatory responses. [ 19 ] It has been found that persistent activation of MAPK8/JNK (representing MAPK8 or JNK family proteins) is closely related to apoptosis and it has been proved that MAPK8/JNK is pro-apoptotic molecule that activates pro-apoptotic genes BAX and BAD and inhibit the activity of anti-apoptotic protein BCL-2 and BCL2L1, thus promoting the of apoptosis. [ 20 ] MAPK8/JNK also plays an important regulatory role in autophagy signaling pathways. MAPK8/JNK activates autophagy-related gene expression, such as ATG5, ATG7 and Beclin1, by phosphorylating its downstream transcription factors AP1. [ 21 ] Whether TNF-α- induced autophagy or caspase-8 inhibitor - induced autophagy, MAPK8/JNK activation and MAPK8/JNK regulated gene expression is essential for autophagy pathway activation. [ 22 ] In the cells necroptosis, MAPK8/JNK is mainly involved in its downstream pathway regulation. After RIPK1 and RIPK3 forming complexes, the MAPK8/JNK is activated to promote downstream pathway transduction of necroptosis. MAPK8/JNK inhibitor can reduce the degree of necroptosis of cells and can alleviate mitochondrial depolarization as well as reduce ROS production. [ 23 ] To identify novel targets for programmed cell death in tumor cells, we performed the drug repurposing strategy by using a high-throughput screen of a FDA-approved compound library, which contains 1068 small molecule drugs with were well studied in biological activity, targets, safetyprofile and bioavailability in various clinical trials. In this study, Menadione was selected and identified as the potential compound which contribute to the colon cancer cells death. Menadione induced various types of cell death by regulating the expression of MAPK8. Therefore, we identified Menadione, a FDA approved drug, may also act as a potential anti-cancer compound by targeting MAPK8. Material And Methods Cell Lines L929 cells, HT29 cells and SW620 cells were cultured in DMEM. All the media were supplemented with 10% fetal bovine serum (v/v), and 100 U·ml − 1 of penicillin/streptomycin. Cells grown under 5% CO 2 at 37°C. Reagents The following reagents were used for induction of cell necroptosis: Mouse Tumor Necrosis Factor ɑ (mTNF-ɑ, 5178SC), Human Tumor Necrosis Factor-ɑ (hTNF-ɑ, 8902SC) were purchased from Cell Signaling Technology; SMAC (T6007), zVAD-fmk (T6013) were obtained from Topscience; and Cycloheximide (HY-12320) was obtained from MCE. FDA approved drug library (L4200) and necrostatin-1 (T1847) were used for drug screening and subsequent experiments. Cell Viability Assay TNFɑ (20 ng/mL) was used to induced necroptosis in L929 cells. TNFɑ (20 ng/mL) plus SMAC (10 nM) or cycloheximide (5 µg/mL) and zVAD-fmk (20 µM) were used to induced necroptosis in MDF and HT29 cells. The drugs were incubated with cells exposed to the above combinations at indicated concentrations for indicated time. CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) was used to examine cell viability. Qrt Pcr And Pcr Array For Programmed Cell Death An Human Signal Transduction PathwayFinder™ RT2 Profiler™ PCR Array (PAHS-014Z, Qiagen, Frederick, MD., USA) was used to screen a panel of 84 genes representative of ten different signal transduction pathways in HT-29 cell. The first-strand cDNA was mixed with 2 × RT2 SYBR Green qPCR Master Mix and ddH2O. The qPCR was performed on an Applied Biosystems (ABI) 7500 according to the RT2 Profiler PCR Array instructions under the following conditions: 95°C for 10 min, then 40 cycles at 95°C for 15 sec and 60°C for 1 min. Each array contained five separate housekeeping genes that were used for normalization of the sample data. Microarray data was normalized against the house keeping genes by calculating the ΔCt for each gene of interest in the plate. Fold changes of gene expression, scatterplot and heatmap were analyzed and generated by using RT2 PCR array data analysis web portal version 3.5 ( http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php ). [ 24 ] Genes of Menadione-treated groups that had fold changes of more than two in expression against negative and positive control groups were considered significant. The candidate genes were chosen to be validated in an additional experiment. Cells were treated with MENA for 1h and treated with tsz for 20-24h. The RNA was then extracted by hot phenol method and further treated with RT reagent with gDNA Eraser (Takara #RR047A, Beijing, China). The PCR was performed using Universal SYBR Green Supermix (Bio-Rad #1725121) with the following program: 95°C for 30 s; 95°C for 5 s; and 60°C for 30 s, for 40 cycles. [ 25 ] Western Blot Analysis Western Blot Analysis Cells were lysed with RIPA lysis buffer containing a phosphatase inhibitor and PMSF. Extracted proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked with 5% (w/v) skim milk powder in TBST for 1 h, and incubated with primary antibodies at 4°C overnight, according to the manufacturer’s protocol (dilution ratio of all proteins was 1:1000). After washing, membranes were incubated with peroxidase-conjugated secondary antibodies (dilution ratio was 1:1000), and protein bands were visualized using the hypersensitive enhanced chemiluminescence (ECL) chemiluminescence kit. Densitometric analysis was performed using the Image J software (1.8.0, NIH, US), and the intensity of specific bands was normalized to the β-actin band. [ 26 ] Functional Enrichment Analysis Gene Ontology (GO) [ 27 ] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [ 28 ] functional and pathway enrichment analysis were performed for mRNAs in prognosis-related co-expression RNA network using the Database for Annotation, Visualization and Integrated Discovery bioinformatics resources (DAVID) ( https://david-d.ncifcrf.gov/ ), and P < 0.05 was considered as the cut-off criterion to screen the Enriched terms and pathways. Establishment Of Protein–protein Interaction (Ppi) Network To understand the underlying interaction of DEmRNAs, the STRING website was employed to construct the PPI network, which was visualized by the Cytoscape software v3.6.1. Statistical Analysis All data in graphs were presented as the mean value ± standard deviation from three independent measurements. The statistical analysis was used in statistical software (SPSS, Chicago, IL, USA) and GraphPad Prism 7.00 (GraphPad Software, CA, USA). p < 0.05 was considered significant. [ 29 ] Result High Throughput Screen to Identify the effect of Menadione in programmed cell death Fas-associated death domain (FADD) is associated with the impediment of various cellular pathways, including apoptosis and necroptosis. [ 30 ] To screen the effective compounds for programmed cell death, we used L929 FADD knockout (L929-FADD-KO) cells as programmed death pathway-sensitive cells. In the high throughput screening, 1,068 compounds from the FDA approved drug library were applied to evaluate the cell viability. Briefly, using a CellTiter-Glo kit to detect the cell viability after treated with compounds and drugs (10µM) in L929-FADD-KO or L929 wild type lines (Fig. 1 A). To screen the most potent and selective compounds, positive hits were defined as the survival rate of the three porous cells treated with the same drug was less than 50% (below 0.5) as compared with untreated cells. The primary screen resulted in the identification of 17 compounds that showed at least 50% lethal for L929-FADD-KO cells (Fig. 1 B). To further explore the contribution of candidate drugs in necroptosis, we treated cells with TNF-α as a positive control to induce cells necropotosis and performed the nec-1 rescue experiment. [ 31 ] Nec-1, a type of alkaloid with small molecule, was first identified as an inhibitor of necrotic cell death by specific blocking RIP1. [ 32 ] In this study, the necrosis inhibitor Nec-1(30µM) was used to rescue necroptosis cell induced either by FDA drugs (10µM) or TNF-α. We used these 17 candidate drugs to screened in L929-FADD-KO (Fig. 1 C) cells and L929 cells (Fig. 1 D) for extensive validation, identifying the lethality of these drugs in different cell lines. As shown, in L929-FADD-KO cells, various drugs could act as cell death agonists for necroptosis (Fig. 1 C). However, in L929 cells, only drug Menadione Evans blue, Crystal Violet, Ponatinib, Ceritinib could be slightly rescued by Nec-1. After an independent sample T test of two sets of data, the cell survival rate of the drug groups with Menadione (P = 0.034) and 10 (panatinib) (P = 0.041) showed the statistical significance. Menadione (Vitamin K3, 2-Methyl-1, 4-naphthoquinone) a synthetic analogue of vitamin K, acts as a provitamin that is converted into a vitamin in the body. Recently, it was reported that Menadione has anti-cancer activity. [ 33 ] Optimization And Comparison Of Treatment Conditions For Menadione In Ht29 Although Menadione was well identified to induce necroptosis in L929 and L929-FADD-KO, the mouse fibroblast cells L929 showed less significant in clinical application. Next, we evaluated the effect of Menadione in colorectal cancer cells, HT-29, to explore its antitumor potential. We first evaluated the effect of the classical cell death combination TSZ, the TNF-α, Smac mimetics and Z-VAD-FMK, [ 34 ] as the positive control in HT-29 (Fig. 2 A). After exploring the concentration of the positive control compounds, we next evaluated the cytotoxicity of Menadione in HT-29. The HT-29 cells were treated with 8µM of Menadione and TSZ (TNF-α:10 ng/mL; Smacmetics :100 nm; Z-VAD-FMK :20µM), and the cell viability was measured at 0.5 h,2h,5h,7h and 28 h. As shown in Fig. 2 B, both of the toxic effect of Menadione and TNF-α increases with the time of action. Besides that, we also conducted the optimal concentration for TSZ and Menadione in HT-29, respectively. The gradient concentration of Menadione was used to treat with HT-29 cells for 24 h. Cell viability was determined using a CellTiter-Glo luminescent cell assay kit (Fig. 2 C, Fig. 2 D,). Menadione can indeed induce HT-29 cell death along with the efficiency increasing concentration. Mechanism Of Ht-29 Cell Death Pathway Induced By Menadione To further determine the potential mechanisms of Menadione in HT-29 cell death, we conducted the array to evaluated the programmed death associating genes regulated by Menadione. The expression of genes related to cells death was determined by high throughput RT²Profiler™PCR Array Human Cell Death Pathway Finder(QIAGEN). The experiment consists of four groups: Control, Menadione-3µM, Menadione-8µM and TSZ. Each group measured 89 genes, followed by data analysis using RPLPO as internal reference genes to screen differentially expressed genes (DEGs). The screening criteria were Fold Change > 2 or Fold Change<-2. A total of 38 genes (24 upregulated genes,14 downregulated genes) were selected from the Menadion-3µM group. A total of 27 genes (16 up-regulated genes,11 down-regulated genes) were selected from the Menadionw-8µM group. A total of 31 genes (22 up-regulated genes, 9 down-regulated genes) were selected from the TSZ group. 15 genes were selected from three groups of Menadione-3µM, Menadione-8µM and TSZ, and the heat map was drawn according to the amount of gene expression, as shown in Fig. 3 A. We can see that there are differences between the genes activated by Menadione and the positive control TSZ, indicating that the pathway of Menadione activation is not exactly the same as the TNF-α, which further verifies the previous view (Fig. 3 A). Menadione could induce cell death and activate programmed death-related pathways in colorectal cancer cells. Different genes are divided into apoptotic group, autophagy group through different death modes regulated by them. The specific values are shown in Fig. 3 B and C. From the histogram, we can intuitively see that in two groups of different cell programmed death patterns, MAPK8 was significantly upregulated in the Menadione treated groups, and with no significant expression change in the TSZ treated group. It suggests that MAPK8 may be a key molecule for Menadione inducing programmed death of HT-29. Identification Of Differentially Expressed Genes And Functional Enrichment Analysis To better understand the potential effects of these dysregulation genes (|fold change| ≥2.0, FDR < 0.05) in Menadione modulated colon cancer, we investigated functional enrichment analysis in Menadione and control samples after PCR array determine. GO analysis results by DAVID database showed the Gene Ontology enrichment analysis (GO enrichment analysis) and KEGG pathway enrichment analysis of 47 differentially expressed genes. The PCR array results demonstrated that Menadione induced programmed death of colon tumor cell is TNF independent cascades. To further explore the mechanisms of the Menadione associating pathway, we selected 47 different expression genes from PCR array data and determined by Go enrichment analysis. GO enrichment analysis was shown to illustrate the functions of cell death associating genes: biological processes (biological process, BP), molecular functions (Molecular function, MF), and cellular components (cellular component, CC). The results of GO enrichment analysis were screened according to P-Value < 0.05, visualized by R software0 (Fig. 4 A). KEGG pathway enrichment analysis were screened by the different expression genes (DEG), and the top20 of KEGG pathway enrichment genes was selected (Fig. 4 B). As shown, the bubble color reflects P-Value, and the bubble size indicates the number of enriched genes in the pathway. Furthermore, we analyzed the protein-protein interaction network (PPI network) in Menadione treatment group (Fig. 4 C), as well as the TSZ treatment group (Fig. 4 D). The proteins were selected by String website ( https://string-db.org/ ) and the PPI networks were visualized by Cytoscape software. Comparing with the PPI of TSZ treatment group, the PPI of Menadione treatment group showed significantly upregulation of MAPK8 and Caspase3. Menadione Regulated Cell Death By Regulation Of Mapk8 Cascades Since the Menadione can cause programmed cell death in HT-29 colorectal cancer cells, which may be mainly involved in apoptosis and necroptosis related pathways, especially regulating by MAPK8. To evaluate the regulation of MAPK8 cascades in Menadione induced programmed cell death, the related molecules were further determined by real-time fluorescence quantitative PCR (RT-qPCR) and western blot (WB). Firstly, we evaluated the regulation of necroptosis associating genes after Menadione (3um and 8um) treatments as well as the TSZ control, respectively. We found both the Menadione (Fig. 5 A) and TSZ (Fig. 5 B) could induce cell necrosis by upregulating MLKL and RIK3. Interestingly, Menadione could significantly increase the expression of MAPK8, caspase 3 and 8 compare with TSZ treatment (Fig. 5 C and D). Our data implied that MAPK8 cascades may be involved in the induction of cell programmed death by a novel Menadione. Discussion In the present study, we screened small molecular compounds from FDA approved drug library to identify the drugs for tumor cell programmed death regulation. After screening with 1068 compounds, we found the Menadione can induce cell death by regulating MAPK8. With the results of bioinformatics analysis including PPI analysis and Go enrichment analysis, we speculated that MAPK8 is a key signal molecule for cell death induced by Menadione. MAPK8 also up-regulated the cell death associating genes in the Menadione treatment group. Interesting, in traditional TSZ (TNF-α, Smac mimetics and Z-VAD-FMK) induced cell death group, nearly no change in MAPK8 expression. These results suggested that MAPK8 may be a key molecule in the differences between the regulatory mechanisms. Nowadays, tumor necrosis factor (TNF) is a kind of cytokines which can cause hemorrhagic necrosis in many kinds of tumors. TNF activates a variety of programmed cell death pathways, including apoptosis and necroptosis. [ 35 ] Although TNF has powerful tumor cell killing effects, when a large number of TNF enter the circulatory system, they induce sepsis-like symptoms, even toxic shock, and ultimately lead to multiple organ failure and death. [ 36 ] Taking into account the limitations of TNF application, it has become one of the new research directions to find some compounds that can replace TNF induce programmed cell death. Currently common drugs for chemotherapy usually play antitumor effects by inducing apoptosis. [ 37 ] However this tumor treatment strategy often leads to severe resistance at later stages, the search for new antitumor agents and programmed death by inducing cells in other ways may become a future drug development strategy, such as looking for TNF-a replacement drugs with fewer side effects. [ 38 ] Therefore, various manners for drug repurposing have been developed. The FDA approved "old medicine" has been used in clinical, what side effects is very clear. Compared with "new drugs", drug repurposing is more safe and reliable, more importantly, " old medicine "also has the huge advantage of saving cost and short research and development cycle. It is considered to be one of the fastest and most effective strategies in new drug development. [ 39 ] Menadione, a essential nutrient often associated with the clotting cascade. [ 40 ] The traditional biological function of Menadione is considered to be maintaining healthy blood clotting and preventing hemorrhageand excessive bleeding. Specifically, it is necessary for the carboxylation of proteins governing the conversion of the blood clotting agent prothrombin to thrombin. [ 41 ] In recent years, many studies have shown that Menadione has a significant inhibitory effect on the development and migration of tumors. [ 42 ] Menadione is thought to play an anti-tumor role by driving substantial ROS production specifically by tumor cells, leading to severe mitochondrial oxidative stress and causing mitochondria-dependent apoptosis in tumor cells. [ 43 ] Besides, Menadione also suppressed invasion, migration and epithelial-mesenchymal transition in human CRC cells by reducing Wnt signaling pathway related gene expression [ 44 ] . However, in our study, we preliminarily explored and discovered possible novel pathways for Menadione to exert anti-tumor effects that completely different from the above. According to our study, Menadione may induce mitochondrial-independent apoptosis in tumor cells by upregulation of MAPK8 expression. This provides new ideas for the future application of Menadione in the antitumor field. For tumor cells that are not sensitive to oxidative stress, Menadione may also play an antitumor role by inducing mitochondrial-independent apoptosis in tumor cells. Declarations -Ethics approval and consent to participate The study was approved by the insititutional review board (CWO) of Shanghai Jiao Tong University -Consent for publication Not applicable. -Availability of data and materials All data generated or analysed during this study are included in this published article. -Competing interests The authors declare that they have no conflicts of interest with the contents of this article. -Funding : This work was sponsored by grants from the National Nature Science Foundation (82173543 and 81803269), Shanghai Municipality Health Commission (GWV-10.2-YQ17) and the Major Science and Technology Innovation Program ofShanghai Municipal Education Commission （2019-01-07-00-01-E00059） -Authors' contributions : For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, Wei Mu.; methodology, Weiwei Song and Liyuan Cao; software, Pengfei Gu and Lei Ding; validation, Weiwei Song and Liyuan Cao; formal analysis, Weiwei Song; investigation, Yanan Wei, Xueqi Ma; resources, Wei Mu; data curation, Weiwei Song; writing—original draft preparation, Weiwei Song; writing—review and editing, Wei Mu.; visualization, Jinli Sun; supervision, Wei Mu and Jinli Sun; project administration, Wei Mu and Jinli Sun; funding acquisition, Wei Mu. All authors have read and agreed to the published version of the manuscript.” Please turn to the CRediT taxonomy for the term explanation. Authorship must be limited to those who have contributed substantially to the work reported. -Acknowledgements We appreciated Yanan Wei for preliminary experiments for the manuscript. References [1] Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C, Norris A, Sanseau P, Cavalla D, Pirmohamed M. Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov. 2019 Jan;18(1):41-58. doi: 10.1038/nrd.2018.168. Epub 2018 Oct 12. PMID: 30310233. [2] Sleire L, Førde HE, Netland IA, Leiss L, Skeie BS, Enger PØ. Drug repurposing in cancer. Pharmacol Res. 2017 Oct;124:74-91. doi: 10.1016/j.phrs.2017.07.013. Epub 2017 Jul 13. PMID: 28712971. [3] Kumar A, Singh UK, Kini SG, Garg V, Agrawal S, Tomar PK, Pathak P, Chaudhary A, Gupta P, Malik A. JNK pathway signaling: a novel and smarter therapeutic targets for various biological diseases. Future Med Chem. 2015;7(15):2065-86. doi: 10.4155/fmc.15.132. Epub 2015 Oct 27. PMID: 26505831. [4] Rosen AW, Degett TH, Gögenur I. [Individualized treatment of colon cancer]. Ugeskr Laeger. 2016 Aug 1;178(31):V11150916. Danish. PMID: 27506915. [5] Fotheringham S, Mozolowski GA, Murray EMA, Kerr DJ. Challenges and solutions in patient treatment strategies for stage II colon cancer. Gastroenterol Rep (Oxf). 2019 Jun;7(3):151-161. doi: 10.1093/gastro/goz006. Epub 2019 Mar 11. PMID: 31217978; PMCID: PMC6573795. [6] Jonas S, Wild C, Schamberger C. "Screening" in speziellen Situationen. Prädiktive humangenetische Diagnostik bei hereditärem Mamma- und Kolorektalkarzinom ["Screening" in special situations. Assessing predictive genetic screening for hereditary breast and colorectal cancer]. Z Arztl Fortbild Qualitatssich. 2003 Feb;97(1):67-71. German. PMID: 12669692. [7] Neuwirtová R. Povede poznávání mechanizmu apoptózy k novým terapeutickým postupům? [Will knowledge of the mechanisms of apoptosis lead to new therapeutic procedures?]. Cas Lek Cesk. 2001 Aug 2;140(15):460-4. Czech. PMID: 11569166. [8] Ucker DS, Levine JS. Exploitation of Apoptotic Regulation in Cancer. Front Immunol. 2018 Feb 27;9:241. doi: 10.3389/fimmu.2018.00241. PMID: 29535707; PMCID: PMC5835066. [9] Huerta S, Goulet EJ, Livingston EH. Colon cancer and apoptosis. Am J Surg. 2006 Apr;191(4):517-26. doi: 10.1016/j.amjsurg.2005.11.009. PMID: 16531147. [10] Dunbar JP, Sulpice R, Dugon MM. The kiss of (cell) death: can venom-induced immune response contribute to dermal necrosis following arthropod envenomations? Clin Toxicol (Phila). 2019 Aug;57(8):677-685. doi: 10.1080/15563650.2019.1578367. Epub 2019 Feb 26. PMID: 30806093. [11] Patel HJ, Patel BM. TNF-α and cancer cachexia: Molecular insights and clinical implications. Life Sci. 2017 Feb 1;170:56-63. doi: 10.1016/j.lfs.2016.11.033. Epub 2016 Dec 3. PMID: 27919820. [12] Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C, Norris A, Sanseau P, Cavalla D, Pirmohamed M. Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov. 2019 Jan;18(1):41-58. doi: 10.1038/nrd.2018.168. Epub 2018 Oct 12. PMID: 30310233. [13] Parvathaneni V, Kulkarni NS, Muth A, Gupta V. Drug repurposing: a promising tool to accelerate the drug discovery process. Drug Discov Today. 2019 Oct;24(10):2076-2085. doi: 10.1016/j.drudis.2019.06.014. Epub 2019 Jun 22. PMID: 31238113. [14] Roskoski R Jr. Properties of FDA-approved small molecule protein kinase inhibitors. Pharmacol Res. 2019 Jun;144:19-50. doi: 10.1016/j.phrs.2019.03.006. Epub 2019 Mar 13. PMID: 30877063. [15] Sleire L, Førde HE, Netland IA, Leiss L, Skeie BS, Enger PØ. Drug repurposing in cancer. Pharmacol Res. 2017 Oct;124:74-91. doi: 10.1016/j.phrs.2017.07.013. Epub 2017 Jul 13. PMID: 28712971. [16] Conrad M, Angeli JP, Vandenabeele P, Stockwell BR. Regulated necrosis: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 2016 May;15(5):348-66. doi: 10.1038/nrd.2015.6. Epub 2016 Jan 18. PMID: 26775689; PMCID: PMC6531857. [17] Wang S, Zhou D, Xu Z, Song J, Qian X, Lv X, Luan J. Anti-tumor Drug Targets Analysis: Current Insight and Future Prospect. Curr Drug Targets. 2019;20(11):1180-1202. doi: 10.2174/1389450120666190402145325. PMID: 30947670. [18] Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000 Oct 13;103(2):239-52. doi: 10.1016/s0092-8674(00)00116-1. PMID: 11057897. [19] Uitdehaag JC, Verkaar F, Alwan H, de Man J, Buijsman RC, Zaman GJ. A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets. Br J Pharmacol. 2012 Jun;166(3):858-76. doi: 10.1111/j.1476-5381.2012.01859.x. PMID: 22250956; PMCID: PMC3417414. [20] Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002 Dec 6;298(5600):1911-2. doi: 10.1126/science.1072682. PMID: 12471242. [21] Yao RQ, Ren C, Xia ZF, Yao YM. Organelle-specific autophagy in inflammatory diseases: a potential therapeutic target underlying the quality control of multiple organelles. Autophagy. 2021 Feb;17(2):385-401. doi: 10.1080/15548627.2020.1725377. Epub 2020 Feb 12. PMID: 32048886; PMCID: PMC8007140. [22] Chen J, Ye C, Wan C, Li G, Peng L, Peng Y, Fang R. The Roles of c-Jun N-Terminal Kinase (JNK) in Infectious Diseases. Int J Mol Sci. 2021 Sep 6;22(17):9640. doi: 10.3390/ijms22179640. PMID: 34502556; PMCID: PMC8431791. [23] Jia Y, Wang F, Guo Q, Li M, Wang L, Zhang Z, Jiang S, Jin H, Chen A, Tan S, Zhang F, Shao J, Zheng S. Curcumol induces RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling in hepatic stellate cells. Redox Biol. 2018 Oct;19:375-387. doi: 10.1016/j.redox.2018.09.007. Epub 2018 Sep 7. PMID: 30237126; PMCID: PMC6142373. [24] Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ. 2005 Sep;29(3):151-9. doi: 10.1152/advan.00019.2005. PMID: 16109794. [25] Yue Y, Zhang Q, Wu S, Wang S, Cui C, Yu M, Sun Z. Identification of key genes involved in JAK/STAT pathway in colorectal cancer. Mol Immunol. 2020 Dec;128:287-297. doi: 10.1016/j.molimm.2020.10.007. Epub 2020 Nov 25. PMID: 33248399. [26] Hnasko TS, Hnasko RM. The Western Blot. Methods Mol Biol. 2015;1318:87-96. doi: 10.1007/978-1-4939-2742-5_9. PMID: 26160567. [27] Hinderer EW 3rd, Moseley HNB. GOcats: A tool for categorizing Gene Ontology into subgraphs of user-defined concepts. PLoS One. 2020 Jun 11;15(6):e0233311. doi: 10.1371/journal.pone.0233311. PMID: 32525872; PMCID: PMC7289357. [28] Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017 Jan 4;45(D1):D353-D361. doi: 10.1093/nar/gkw1092. Epub 2016 Nov 28. PMID: 27899662; PMCID: PMC5210567. [29] Andrade C. The P Value and Statistical Significance: Misunderstandings, Explanations, Challenges, and Alternatives. Indian J Psychol Med. 2019 May-Jun;41(3):210-215. doi: 10.4103/IJPSYM.IJPSYM_193_19. PMID: 31142921; PMCID: PMC6532382. [30] Lee EW, Seo J, Jeong M, Lee S, Song J. The roles of FADD in extrinsic apoptosis and necroptosis. BMB Rep. 2012 Sep;45(9):496-508. doi: 10.5483/bmbrep.2012.45.9.186. PMID: 23010170. [31] Sosna J, Voigt S, Mathieu S, Lange A, Thon L, Davarnia P, Herdegen T, Linkermann A, Rittger A, Chan FK, Kabelitz D, Schütze S, Adam D. TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death. Cell Mol Life Sci. 2014 Jan;71(2):331-48. doi: 10.1007/s00018-013-1381-6. Epub 2013 Jun 13. PMID: 23760205; PMCID: PMC3889832. [32] Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. doi: 10.1038/nchembio711. Epub 2005 May 29. Erratum in: Nat Chem Biol. 2005 Sep;1(4):234. PMID: 16408008. [33] Popa DS, Bigman G, Rusu ME. The Role of Vitamin K in Humans: Implication in Aging and Age-Associated Diseases. Antioxidants (Basel). 2021 Apr 6;10(4):566. doi: 10.3390/antiox10040566. PMID: 33917442; PMCID: PMC8067486. [34] He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009 Jun 12;137(6):1100-11. doi: 10.1016/j.cell.2009.05.021. PMID: 19524512. [35] Holbrook J, Lara-Reyna S, Jarosz-Griffiths H, McDermott M. Tumour necrosis factor signalling in health and disease. F1000Res. 2019 Jan 28;8:F1000 Faculty Rev-111. doi: 10.12688/f1000research.17023.1. PMID: 30755793; PMCID: PMC6352924. [36] Sfikakis PP. The first decade of biologic TNF antagonists in clinical practice: lessons learned, unresolved issues and future directions. Curr Dir Autoimmun. 2010;11:180-210. doi: 10.1159/000289205. Epub 2010 Feb 18. PMID: 20173395. [37] Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D'Orazi G. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging (Albany NY). 2016 Apr;8(4):603-19. doi: 10.18632/aging.100934. PMID: 27019364; PMCID: PMC4925817. [38] Mohammad RM, Muqbil I, Lowe L, Yedjou C, Hsu HY, Lin LT, Siegelin MD, Fimognari C, Kumar NB, Dou QP, Yang H, Samadi AK, Russo GL, Spagnuolo C, Ray SK, Chakrabarti M, Morre JD, Coley HM, Honoki K, Fujii H, Georgakilas AG, Amedei A, Niccolai E, Amin A, Ashraf SS, Helferich WG, Yang X, Boosani CS, Guha G, Bhakta D, Ciriolo MR, Aquilano K, Chen S, Mohammed SI, Keith WN, Bilsland A, Halicka D, Nowsheen S, Azmi AS. Broad targeting of resistance to apoptosis in cancer. Semin Cancer Biol. 2015 Dec;35 Suppl(0):S78-S103. doi: 10.1016/j.semcancer.2015.03.001. Epub 2015 Apr 28. PMID: 25936818; PMCID: PMC4720504. [39] Sleire L, Førde HE, Netland IA, Leiss L, Skeie BS, Enger PØ. Drug repurposing in cancer. Pharmacol Res. 2017 Oct;124:74-91. doi: 10.1016/j.phrs.2017.07.013. Epub 2017 Jul 13. PMID: 28712971. [40] Lamson DW, Plaza SM. The anticancer effects of vitamin K. Altern Med Rev. 2003 Aug;8(3):303-18. PMID: 12946240. [41] Semkova S, Zhelev Z, Miller T, Sugaya K, Aoki I, Higashi T, Bakalova R. Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin. Anticancer Res. 2020 Apr;40(4):1963-1972. doi: 10.21873/anticanres.14151. PMID: 32234885. [42] Calderon PB, Cadrobbi J, Marques C, Hong-Ngoc N, Jamison JM, Gilloteaux J, Summers JL, Taper HS. Potential therapeutic application of the association of vitamins C and K3 in cancer treatment. Curr Med Chem. 2002 Dec;9(24):2271-85. doi: 10.2174/0929867023368674. PMID: 12470246. [43] Semkova S, Zhelev Z, Miller T, Sugaya K, Aoki I, Higashi T, Bakalova R. Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin. Anticancer Res. 2020 Apr;40(4):1963-1972. doi: 10.21873/anticanres.14151. PMID: 32234885. [44] Kishore C, Sundaram S, Karunagaran D. Vitamin K3 (Menadione) suppresses epithelial-mesenchymal-transition and Wnt signaling pathway in human colorectal cancer cells. Chem Biol Interact. 2019 Aug 25;309:108725. doi: 10.1016/j.cbi.2019.108725. Epub 2019 Jun 22. PMID: 31238027. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1406834","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":88918226,"identity":"df9834b9-0ba7-473e-b2d3-8b07df6bc24a","order_by":0,"name":"Weiwei Song","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Song","suffix":""},{"id":88918227,"identity":"2fb6a913-1d76-41d8-9fae-e69932100ab0","order_by":1,"name":"Liyuan Cao","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liyuan","middleName":"","lastName":"Cao","suffix":""},{"id":88918229,"identity":"6d2a4f7f-cbd5-4ba7-8c4a-e49fb4febcaa","order_by":2,"name":"Lei Ding","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Ding","suffix":""},{"id":88918232,"identity":"3fb18238-0f7d-402c-997d-1c299cc67062","order_by":3,"name":"Xueqi Ma","email":"","orcid":"","institution":"Shanghai Institute of Nutrition and Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xueqi","middleName":"","lastName":"Ma","suffix":""},{"id":88918234,"identity":"c494e096-73da-44bc-83e6-93f9cc890a8a","order_by":4,"name":"Pengfei Gu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Gu","suffix":""},{"id":88918235,"identity":"8b57e152-f312-4cdf-a7d9-bfcba600cd8c","order_by":5,"name":"Wenbo Wang","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenbo","middleName":"","lastName":"Wang","suffix":""},{"id":88918236,"identity":"adfb1e1f-d919-4988-8ac6-c3f51f9088f4","order_by":6,"name":"Jinli Sun","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinli","middleName":"","lastName":"Sun","suffix":""},{"id":88918238,"identity":"158168ec-9516-4a4e-910a-a2b690248c5d","order_by":7,"name":"Wei Mu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFACxgYGhgqGBAiHjWgtZ0jTAtLVRooWg+PNrZt559XlyU87Y8DwoewwA//sBgJazhxsu8277XCxwe0cA8YZ5w4zSNw5gF+L2Y1EkJYDiRukcwyYedsOMxhIJBDQcv8hUMucusT5s4Fa/hKl5QYjUEsDc2ID0GHMjMRosT+T2HZzzrHDiRtupxUc7DmXziNxg4AWyfbjz268qQE5LHnjgx9l1nL8MwhoQQEHgJiHBPWjYBSMglEwCnABACMHSLxKWLtkAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Mu","suffix":""}],"badges":[],"createdAt":"2022-03-01 07:29:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1406834/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1406834/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19090620,"identity":"7f193eb9-1f52-45a6-a99a-711ccf738d55","added_by":"auto","created_at":"2022-03-10 19:04:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":569514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of FDA approved drugs in L929 and L929-FADD-KO cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A) Strategy for drug screening of inhibiting necroptosis. (B, C) Identification of 17 candidate drugs from FDA approved libraries in L929 cells. L929 cells were treated with TNFɑ(20ng/mL) and each drug(10µM) for 24hr. L929 cells treated with TNFɑ(20ng/mL) and necrostatin-1 (10µM) were regarded as the positive control. (D) Verification of 17 candidate drugs in L929-FADD-KO cells. Cells were treated with 10µM candidate drugs (or nec-1) for 8hr. The data are represented as the mean ± SEM of triplicate wells. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-1406834/v1/bb0eb64ed4e477d4f01398cd.png"},{"id":19090616,"identity":"ae6245ca-be04-478c-8cab-3f3bf3f4aa95","added_by":"auto","created_at":"2022-03-10 19:04:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization and comparison of treatment conditions for Menadione \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A, B) the toxic effect of Menadione and TNF-α with different treatment time in HT-29. (C, D) the toxic effect of Menadione and TNF-α with different concentration in HT-29.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-1406834/v1/ea6098d27a1f50fdd80d2837.png"},{"id":19090617,"identity":"f42cc0cb-3b66-44fe-bee4-21bccac9e28d","added_by":"auto","created_at":"2022-03-10 19:04:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":209195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanisms of programmed cell death regulated by Menadione\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A) Heat map of transcripts analyzed by PCR array. Expression values were normalized over the expression of GAPDH and presented as log10 of relative changes. As shown, genes with higher expression are depicted in red, genes with lower expression are depicted in green. (B,C) Expression of programmed cell death associating genes by qRT PCR.\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-1406834/v1/49d1d0cb89d4c5bc54c8a8c6.png"},{"id":19090872,"identity":"3bf9b0ae-4e48-4ec2-aac7-e00d95bf0eb1","added_by":"auto","created_at":"2022-03-10 19:07:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1174857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Menadione induced genes and pathways\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A) Gene Ontology (GO) analysis of DEGs. Go annotation of the domains of biological processes, cellular components and molecular functions. Top 20 terms are shown at –log (p-value). (B) The KEGG pathway enrichment analysis of Top 20 terms for DEGs and shown at -log10(p-value). (C) Protein-protein interaction network for DEGs with |fold change| ≥2.0, P \u0026lt; 0.05. Upregulated and downregulated genes are shaded red and green, respectively.\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-1406834/v1/cab1951cbe9d3e19cda47820.png"},{"id":19090619,"identity":"82c0a8b7-a07a-4341-9a81-8cdc5f39fa1e","added_by":"auto","created_at":"2022-03-10 19:04:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":197532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMenadione up-regulated MAPK8 cascades \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eqRT-PCR analysis of necrosis genes in HT-29 with Menadione(A) and TSZ (B) treatments. qRT-PCR(C) analysis and WB (D) of apoptosis genes in HT-29 with Menadione treatments.\u0026nbsp;\u003c/p\u003e","description":"","filename":"FIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-1406834/v1/e624aa8cdc3705e5a31d25d1.png"},{"id":21638921,"identity":"dccb356f-4a0c-49cb-a03b-da3d24453dcd","added_by":"auto","created_at":"2022-05-18 22:29:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2231437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1406834/v1/dc267f1a-3680-47c7-84da-4a30cdcfc302.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eScreening of a FDA-Approved Compound Library Identifies Menadione in Regulating Colon Cancer Programmed Death via MAPK8 Cascades\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e Globally, 1.36\u0026nbsp;million people are affected accounting for nearly 10% of cancer prevalence\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. It remains the second leading cause of cancer worldwide\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The most widely available treatments for managing colon cancer include surgery, radiation therapy, and chemotherapy.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e Although some progress has been made in various of researches and development of CRC drugs in recent years, there are still many limitations and challenges, such as the side effects of chemotherapy and radiation, the limitation of targeted therapy because of KRAS or BRAF gene mutations, and the drug resistance of tumor cells caused by long-term drug medication.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e In spite of their conceptual promise, more than one-third of colon cancer patients are often incurably.\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e With the development of the science, increasing attentions have been paid to the treatment of cancer by inducing the programmed death of tumor cells to regulate tumorigenesis.\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e The implementation of new therapeutic options for the management of colon cancer mandates a revisit to programmed cell death with an emphasis on the mechanisms.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e TNF-α is known to act as a necroptosis inducer.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e However, there are many problems in the application of TNF-α in clinical tumor treatment. For example, multiple organ failure caused by possible inflammatory cytokine storms is a major problem..\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e Thus, developing the novel compounds to induce cell death, exploring the signaling pathways according to molecular basis of colon carcinogenesis and developing new drugs targeting them are necessary.\u003c/p\u003e \u003cp\u003eGiven the high attrition rates, a huge sum of costs and long cycle of discovery and development of new drugs, the use of \"old\" drugs to treat common and rare diseases is gradually becoming an attractive proposition.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e Repurposing well-studied preclinical, clinical, and approved compounds holds the greatest potential to swiftly move a drug candidate from the bench to the clinic.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e An FDA-approved compound library (TargetMol L4200) was well-studied compounds, many of which already possess extensive human safety data.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e For instance, based on FDA drug library, thalidomide has been found to be used in the treatment of systemic lupus erythematosus and multiple myeloma.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e Meanwhile, new drug strategies also could be focused on the regulation of cell death pathways, contributing to a better comprehension of cancer pathogenesis and therapeutics.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e Supportive evidence has increasingly revealed the prime molecular mechanisms of each cell death subroutine and thus providing a succession of possible targets in cancer therapy, w\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehereas\u003c/span\u003e the complicated relationships between different cell death subroutines still remain to be elucidated.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Summary of above mentioned, drug repurposing through screening a FDA-approved drug library via programmed cell death would be the feasible method to develop novel drug for colorectal cancer.\u003c/p\u003e \u003cp\u003eMAPK8(Mitogen-activated JNK1) is a mumber of MAPK family and JNK family encoding by a MAPK8 gene. MAPK family is one of serine-threonine protein kinases family that plays an important role in physiological and pathological process.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e MAPK8 as a member of the MAPK family can activate and phosphorylate transcription factors (such as AP-1), thereby activating downstream genes that play an important regulatory role in cell proliferation, differentiation, death, and inflammatory responses.\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e It has been found that persistent activation of MAPK8/JNK (representing MAPK8 or JNK family proteins) is closely related to apoptosis and it has been proved that MAPK8/JNK is pro-apoptotic molecule that activates pro-apoptotic genes BAX and BAD and inhibit the activity of anti-apoptotic protein BCL-2 and BCL2L1, thus promoting the of apoptosis.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e MAPK8/JNK also plays an important regulatory role in autophagy signaling pathways. MAPK8/JNK activates autophagy-related gene expression, such as ATG5, ATG7 and Beclin1, by phosphorylating its downstream transcription factors AP1.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e Whether TNF-α- induced autophagy or caspase-8 inhibitor - induced autophagy, MAPK8/JNK activation and MAPK8/JNK regulated gene expression is essential for autophagy pathway activation.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e In the cells necroptosis, MAPK8/JNK is mainly involved in its downstream pathway regulation. After RIPK1 and RIPK3 forming complexes, the MAPK8/JNK is activated to promote downstream pathway transduction of necroptosis. MAPK8/JNK inhibitor can reduce the degree of necroptosis of cells and can alleviate mitochondrial depolarization as well as reduce ROS production.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo identify novel targets for programmed cell death in tumor cells, we performed the drug repurposing strategy by using a high-throughput screen of a FDA-approved compound library, which contains 1068 small molecule drugs with were well studied in biological activity, targets, safetyprofile and bioavailability in various clinical trials. In this study, Menadione was selected and identified as the potential compound which contribute to the colon cancer cells death. Menadione induced various types of cell death by regulating the expression of MAPK8. Therefore, we identified Menadione, a FDA approved drug, may also act as a potential anti-cancer compound by targeting MAPK8.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines\u003c/h2\u003e \u003cp\u003eL929 cells, HT29 cells and SW620 cells were cultured in DMEM. All the media were supplemented with 10% fetal bovine serum (v/v), and 100 U\u0026middot;ml\u0026thinsp;\u0026minus;\u0026thinsp;1 of penicillin/streptomycin. Cells grown under 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eReagents\u003c/h2\u003e\n\u003cp\u003eThe following reagents were used for induction of cell necroptosis: Mouse Tumor Necrosis Factor ɑ (mTNF-ɑ, 5178SC), Human Tumor Necrosis Factor-ɑ (hTNF-ɑ, 8902SC) were purchased from Cell Signaling Technology; SMAC (T6007), zVAD-fmk (T6013) were obtained from Topscience; and Cycloheximide (HY-12320) was obtained from MCE. FDA approved drug library (L4200) and necrostatin-1 (T1847) were used for drug screening and subsequent experiments.\u003c/p\u003e\n\u003ch2\u003eCell Viability Assay\u003c/h2\u003e\n\u003cp\u003eTNFɑ (20 ng/mL) was used to induced necroptosis in L929 cells. TNFɑ (20 ng/mL) plus SMAC (10 nM) or cycloheximide (5 \u0026micro;g/mL) and zVAD-fmk (20 \u0026micro;M) were used to induced necroptosis in MDF and HT29 cells. The drugs were incubated with cells exposed to the above combinations at indicated concentrations for indicated time. CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) was used to examine cell viability.\u003c/p\u003e\n\u003ch2\u003eQrt Pcr And Pcr Array For Programmed Cell Death\u003c/h2\u003e\n\u003cp\u003eAn Human Signal Transduction PathwayFinder\u0026trade; RT2 Profiler\u0026trade; PCR Array (PAHS-014Z, Qiagen, Frederick, MD., USA) was used to screen a panel of 84 genes representative of ten different signal transduction pathways in HT-29 cell. The first-strand cDNA was mixed with 2 \u0026times; RT2 SYBR Green qPCR Master Mix and ddH2O. The qPCR was performed on an Applied Biosystems (ABI) 7500 according to the RT2 Profiler PCR Array instructions under the following conditions: 95\u0026deg;C for 10 min, then 40 cycles at 95\u0026deg;C for 15 sec and 60\u0026deg;C for 1 min. Each array contained five separate housekeeping genes that were used for normalization of the sample data. Microarray data was normalized against the house keeping genes by calculating the ΔCt for each gene of interest in the plate. Fold changes of gene expression, scatterplot and heatmap were analyzed and generated by using RT2 PCR array data analysis web portal version 3.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php\u003c/span\u003e\u003c/span\u003e).\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e Genes of Menadione-treated groups that had fold changes of more than two in expression against negative and positive control groups were considered significant. The candidate genes were chosen to be validated in an additional experiment. Cells were treated with MENA for 1h and treated with tsz for 20-24h. The RNA was then extracted by hot phenol method and further treated with RT reagent with gDNA Eraser (Takara #RR047A, Beijing, China). The PCR was performed using Universal SYBR Green Supermix (Bio-Rad #1725121) with the following program: 95\u0026deg;C for 30 s; 95\u0026deg;C for 5 s; and 60\u0026deg;C for 30 s, for 40 cycles. \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blot Analysis\u003c/div\u003e \u003cp\u003eCells were lysed with RIPA lysis buffer containing a phosphatase inhibitor and PMSF. Extracted proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked with 5% (w/v) skim milk powder in TBST for 1 h, and incubated with primary antibodies at 4\u0026deg;C overnight, according to the manufacturer\u0026rsquo;s protocol (dilution ratio of all proteins was 1:1000). After washing, membranes were incubated with peroxidase-conjugated secondary antibodies (dilution ratio was 1:1000), and protein bands were visualized using the hypersensitive enhanced chemiluminescence (ECL) chemiluminescence kit. Densitometric analysis was performed using the Image J software (1.8.0, NIH, US), and the intensity of specific bands was normalized to the β-actin band.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eFunctional Enrichment Analysis\u003c/h2\u003e\n\u003cp\u003eGene Ontology (GO)\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e and Kyoto Encyclopedia of Genes and Genomes (KEGG)\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e functional and pathway enrichment analysis were performed for mRNAs in prognosis-related co-expression RNA network using the Database for Annotation, Visualization and Integrated Discovery bioinformatics resources (DAVID) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david-d.ncifcrf.gov/\u003c/span\u003e\u003c/span\u003e), and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as the cut-off criterion to screen the Enriched terms and pathways.\u003c/p\u003e\n\u003ch2\u003eEstablishment Of Protein–protein Interaction (Ppi) Network\u003c/h2\u003e\n\u003cp\u003eTo understand the underlying interaction of DEmRNAs, the STRING website was employed to construct the PPI network, which was visualized by the Cytoscape software v3.6.1.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data in graphs were presented as the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from three independent measurements. The statistical analysis was used in statistical software (SPSS, Chicago, IL, USA) and GraphPad Prism 7.00 (GraphPad Software, CA, USA). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHigh Throughput Screen to Identify the effect of Menadione in programmed cell death\u003c/h2\u003e \u003cp\u003eFas-associated death domain (FADD) is associated with the impediment of various cellular pathways, including apoptosis and necroptosis.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e To screen the effective compounds for programmed cell death, we used L929 FADD knockout (L929-FADD-KO) cells as programmed death pathway-sensitive cells. In the high throughput screening, 1,068 compounds from the FDA approved drug library were applied to evaluate the cell viability. Briefly, using a CellTiter-Glo kit to detect the cell viability after treated with compounds and drugs (10\u0026micro;M) in L929-FADD-KO or L929 wild type lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To screen the most potent and selective compounds, positive hits were defined as the survival rate of the three porous cells treated with the same drug was less than 50% (below 0.5) as compared with untreated cells. The primary screen resulted in the identification of 17 compounds that showed at least 50% lethal for L929-FADD-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the contribution of candidate drugs in necroptosis, we treated cells with TNF-α as a positive control to induce cells necropotosis and performed the nec-1 rescue experiment.\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e Nec-1, a type of alkaloid with small molecule, was first identified as an inhibitor of necrotic cell death by specific blocking RIP1.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e In this study, the necrosis inhibitor Nec-1(30\u0026micro;M) was used to rescue necroptosis cell induced either by FDA drugs (10\u0026micro;M) or TNF-α. We used these 17 candidate drugs to screened in L929-FADD-KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) cells and L929 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) for extensive validation, identifying the lethality of these drugs in different cell lines. As shown, in L929-FADD-KO cells, various drugs could act as cell death agonists for necroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, in L929 cells, only drug Menadione Evans blue, Crystal Violet, Ponatinib, Ceritinib could be slightly rescued by Nec-1. After an independent sample T test of two sets of data, the cell survival rate of the drug groups with Menadione (P\u0026thinsp;=\u0026thinsp;0.034) and 10 (panatinib) (P\u0026thinsp;=\u0026thinsp;0.041) showed the statistical significance. Menadione (Vitamin K3, 2-Methyl-1, 4-naphthoquinone) a synthetic analogue of vitamin K, acts as a provitamin that is converted into a vitamin in the body. Recently, it was reported that Menadione has anti-cancer activity.\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eOptimization And Comparison Of Treatment Conditions For Menadione In Ht29\u003c/h2\u003e\n\u003cp\u003eAlthough Menadione was well identified to induce necroptosis in L929 and L929-FADD-KO, the mouse fibroblast cells L929 showed less significant in clinical application. Next, we evaluated the effect of Menadione in colorectal cancer cells, HT-29, to explore its antitumor potential. We first evaluated the effect of the classical cell death combination TSZ, the TNF-α, Smac mimetics and Z-VAD-FMK,\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e as the positive control in HT-29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After exploring the concentration of the positive control compounds, we next evaluated the cytotoxicity of Menadione in HT-29. The HT-29 cells were treated with 8\u0026micro;M of Menadione and TSZ (TNF-α:10 ng/mL; Smacmetics :100 nm; Z-VAD-FMK :20\u0026micro;M), and the cell viability was measured at 0.5 h,2h,5h,7h and 28 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, both of the toxic effect of Menadione and TNF-α increases with the time of action. Besides that, we also conducted the optimal concentration for TSZ and Menadione in HT-29, respectively. The gradient concentration of Menadione was used to treat with HT-29 cells for 24 h. Cell viability was determined using a CellTiter-Glo luminescent cell assay kit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,). Menadione can indeed induce HT-29 cell death along with the efficiency increasing concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch2\u003eMechanism Of Ht-29 Cell Death Pathway Induced By Menadione\u003c/h2\u003e\n\u003cp\u003eTo further determine the potential mechanisms of Menadione in HT-29 cell death, we conducted the array to evaluated the programmed death associating genes regulated by Menadione. The expression of genes related to cells death was determined by high throughput RT\u0026sup2;Profiler\u0026trade;PCR Array Human Cell Death Pathway Finder(QIAGEN). The experiment consists of four groups: Control, Menadione-3\u0026micro;M, Menadione-8\u0026micro;M and TSZ. Each group measured 89 genes, followed by data analysis using RPLPO as internal reference genes to screen differentially expressed genes (DEGs). The screening criteria were Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;2 or Fold Change\u0026lt;-2. A total of 38 genes (24 upregulated genes,14 downregulated genes) were selected from the Menadion-3\u0026micro;M group. A total of 27 genes (16 up-regulated genes,11 down-regulated genes) were selected from the Menadionw-8\u0026micro;M group. A total of 31 genes (22 up-regulated genes, 9 down-regulated genes) were selected from the TSZ group. 15 genes were selected from three groups of Menadione-3\u0026micro;M, Menadione-8\u0026micro;M and TSZ, and the heat map was drawn according to the amount of gene expression, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. We can see that there are differences between the genes activated by Menadione and the positive control TSZ, indicating that the pathway of Menadione activation is not exactly the same as the TNF-α, which further verifies the previous view (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Menadione could induce cell death and activate programmed death-related pathways in colorectal cancer cells. Different genes are divided into apoptotic group, autophagy group through different death modes regulated by them. The specific values are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C. From the histogram, we can intuitively see that in two groups of different cell programmed death patterns, MAPK8 was significantly upregulated in the Menadione treated groups, and with no significant expression change in the TSZ treated group. It suggests that MAPK8 may be a key molecule for Menadione inducing programmed death of HT-29.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch2\u003eIdentification Of Differentially Expressed Genes And Functional Enrichment Analysis\u003c/h2\u003e\n\u003cp\u003eTo better understand the potential effects of these dysregulation genes (|fold change| \u0026ge;2.0, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Menadione modulated colon cancer, we investigated functional enrichment analysis in Menadione and control samples after PCR array determine. GO analysis results by DAVID database showed the Gene Ontology enrichment analysis (GO enrichment analysis) and KEGG pathway enrichment analysis of 47 differentially expressed genes. The PCR array results demonstrated that Menadione induced programmed death of colon tumor cell is TNF independent cascades. To further explore the mechanisms of the Menadione associating pathway, we selected 47 different expression genes from PCR array data and determined by Go enrichment analysis. GO enrichment analysis was shown to illustrate the functions of cell death associating genes: biological processes (biological process, BP), molecular functions (Molecular function, MF), and cellular components (cellular component, CC). The results of GO enrichment analysis were screened according to P-Value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, visualized by R software0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). KEGG pathway enrichment analysis were screened by the different expression genes (DEG), and the top20 of KEGG pathway enrichment genes was selected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). As shown, the bubble color reflects P-Value, and the bubble size indicates the number of enriched genes in the pathway. Furthermore, we analyzed the protein-protein interaction network (PPI network) in Menadione treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), as well as the TSZ treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The proteins were selected by String website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003c/span\u003e) and the PPI networks were visualized by Cytoscape software. Comparing with the PPI of TSZ treatment group, the PPI of Menadione treatment group showed significantly upregulation of MAPK8 and Caspase3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch2\u003eMenadione Regulated Cell Death By Regulation Of Mapk8 Cascades\u003c/h2\u003e\n\u003cp\u003eSince the Menadione can cause programmed cell death in HT-29 colorectal cancer cells, which may be mainly involved in apoptosis and necroptosis related pathways, especially regulating by MAPK8. To evaluate the regulation of MAPK8 cascades in Menadione induced programmed cell death, the related molecules were further determined by real-time fluorescence quantitative PCR (RT-qPCR) and western blot (WB). Firstly, we evaluated the regulation of necroptosis associating genes after Menadione (3um and 8um) treatments as well as the TSZ control, respectively. We found both the Menadione (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and TSZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) could induce cell necrosis by upregulating MLKL and RIK3. Interestingly, Menadione could significantly increase the expression of MAPK8, caspase 3 and 8 compare with TSZ treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). Our data implied that MAPK8 cascades may be involved in the induction of cell programmed death by a novel Menadione.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we screened small molecular compounds from FDA approved drug library to identify the drugs for tumor cell programmed death regulation. After screening with 1068 compounds, we found the Menadione can induce cell death by regulating MAPK8. With the results of bioinformatics analysis including PPI analysis and Go enrichment analysis, we speculated that MAPK8 is a key signal molecule for cell death induced by Menadione. MAPK8 also up-regulated the cell death associating genes in the Menadione treatment group. Interesting, in traditional TSZ (TNF-α, Smac mimetics and Z-VAD-FMK) induced cell death group, nearly no change in MAPK8 expression. These results suggested that MAPK8 may be a key molecule in the differences between the regulatory mechanisms.\u003c/p\u003e \u003cp\u003eNowadays, tumor necrosis factor (TNF) is a kind of cytokines which can cause hemorrhagic necrosis in many kinds of tumors. TNF activates a variety of programmed cell death pathways, including apoptosis and necroptosis.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e Although TNF has powerful tumor cell killing effects, when a large number of TNF enter the circulatory system, they induce sepsis-like symptoms, even toxic shock, and ultimately lead to multiple organ failure and death.\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e Taking into account the limitations of TNF application, it has become one of the new research directions to find some compounds that can replace TNF induce programmed cell death. Currently common drugs for chemotherapy usually play antitumor effects by inducing apoptosis.\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e However this tumor treatment strategy often leads to severe resistance at later stages, the search for new antitumor agents and programmed death by inducing cells in other ways may become a future drug development strategy, such as looking for TNF-a replacement drugs with fewer side effects.\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e Therefore, various manners for drug repurposing have been developed. The FDA approved \"old medicine\" has been used in clinical, what side effects is very clear. Compared with \"new drugs\", drug repurposing is more safe and reliable, more importantly, \" old medicine \"also has the huge advantage of saving cost and short research and development cycle. It is considered to be one of the fastest and most effective strategies in new drug development.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMenadione, a essential nutrient often associated with the clotting cascade.\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003eThe traditional biological function of Menadione is considered to be maintaining healthy blood clotting and preventing hemorrhageand excessive bleeding. Specifically, it is necessary for the carboxylation of proteins governing the conversion of the blood clotting agent prothrombin to thrombin.\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e In recent years, many studies have shown that Menadione has a significant inhibitory effect on the development and migration of tumors.\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e Menadione is thought to play an anti-tumor role by driving substantial ROS production specifically by tumor cells, leading to severe mitochondrial oxidative stress and causing mitochondria-dependent apoptosis in tumor cells.\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e Besides, Menadione also suppressed invasion, migration and epithelial-mesenchymal transition in human CRC cells by reducing Wnt signaling pathway related gene expression\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. However, in our study, we preliminarily explored and discovered possible novel pathways for Menadione to exert anti-tumor effects that completely different from the above. According to our study, Menadione may induce mitochondrial-independent apoptosis in tumor cells by upregulation of MAPK8 expression. This provides new ideas for the future application of Menadione in the antitumor field. For tumor cells that are not sensitive to oxidative stress, Menadione may also play an antitumor role by inducing mitochondrial-independent apoptosis in tumor cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;-Ethics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the insititutional review board (CWO) of Shanghai Jiao Tong University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Consent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Availability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;-Funding\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was sponsored by grants from the National Nature Science Foundation (82173543 and 81803269), Shanghai Municipality Health Commission (GWV-10.2-YQ17) and the Major Science and Technology Innovation Program ofShanghai Municipal Education Commission （2019-01-07-00-01-E00059）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Authors' contributions\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used \u0026ldquo;Conceptualization, Wei Mu.; methodology, Weiwei Song and Liyuan Cao; software, Pengfei Gu and Lei Ding; validation, Weiwei Song and Liyuan Cao; formal analysis, Weiwei Song; investigation, Yanan Wei, Xueqi Ma; resources, Wei Mu; data curation, Weiwei Song; writing\u0026mdash;original draft preparation, Weiwei Song; writing\u0026mdash;review and editing, Wei Mu.; visualization, Jinli Sun; supervision, Wei Mu and Jinli Sun; project administration, Wei Mu and Jinli Sun; funding acquisition, Wei Mu. All authors have read and agreed to the published version of the manuscript.\u0026rdquo; Please turn to the CRediT taxonomy for the term explanation. Authorship must be limited to those who have contributed substantially to the work reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;-Acknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciated Yanan Wei for preliminary experiments for the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e[1] Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C, Norris A, Sanseau P, Cavalla D, Pirmohamed M. Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov. 2019 Jan;18(1):41-58. doi: 10.1038/nrd.2018.168. Epub 2018 Oct 12. PMID: 30310233.\u003c/p\u003e\n\u003cp\u003e[2] Sleire L, F\u0026oslash;rde HE, Netland IA, Leiss L, Skeie BS, Enger P\u0026Oslash;. Drug repurposing in cancer. Pharmacol Res. 2017 Oct;124:74-91. doi: 10.1016/j.phrs.2017.07.013. Epub 2017 Jul 13. PMID: 28712971.\u003c/p\u003e\n\u003cp\u003e[3] Kumar A, Singh UK, Kini SG, Garg V, Agrawal S, Tomar PK, Pathak P, Chaudhary A, Gupta P, Malik A. JNK pathway signaling: a novel and smarter therapeutic targets for various biological diseases. Future Med Chem. 2015;7(15):2065-86. doi: 10.4155/fmc.15.132. Epub 2015 Oct 27. PMID: 26505831.\u003c/p\u003e\n\u003cp\u003e[4] Rosen AW, Degett TH, G\u0026ouml;genur I. [Individualized treatment of colon cancer]. Ugeskr Laeger. 2016 Aug 1;178(31):V11150916. Danish. PMID: 27506915.\u003c/p\u003e\n\u003cp\u003e[5] Fotheringham S, Mozolowski GA, Murray EMA, Kerr DJ. Challenges and solutions in patient treatment strategies for stage II colon cancer. Gastroenterol Rep (Oxf). 2019 Jun;7(3):151-161. doi: 10.1093/gastro/goz006. Epub 2019 Mar 11. PMID: 31217978; PMCID: PMC6573795.\u003c/p\u003e\n\u003cp\u003e[6] Jonas S, Wild C, Schamberger C. \"Screening\" in speziellen Situationen. Pr\u0026auml;diktive humangenetische Diagnostik bei heredit\u0026auml;rem Mamma- und Kolorektalkarzinom [\"Screening\" in special situations. Assessing predictive genetic screening for hereditary breast and colorectal cancer]. Z Arztl Fortbild Qualitatssich. 2003 Feb;97(1):67-71. German. PMID: 12669692.\u003c/p\u003e\n\u003cp\u003e[7] Neuwirtov\u0026aacute; R. Povede pozn\u0026aacute;v\u0026aacute;n\u0026iacute; mechanizmu apopt\u0026oacute;zy k nov\u0026yacute;m terapeutick\u0026yacute;m postupům? [Will knowledge of the mechanisms of apoptosis lead to new therapeutic procedures?]. Cas Lek Cesk. 2001 Aug 2;140(15):460-4. Czech. PMID: 11569166.\u003c/p\u003e\n\u003cp\u003e[8] Ucker DS, Levine JS. Exploitation of Apoptotic Regulation in Cancer. Front Immunol. 2018 Feb 27;9:241. doi: 10.3389/fimmu.2018.00241. PMID: 29535707; PMCID: PMC5835066.\u003c/p\u003e\n\u003cp\u003e[9] Huerta S, Goulet EJ, Livingston EH. Colon cancer and apoptosis. Am J Surg. 2006 Apr;191(4):517-26. doi: 10.1016/j.amjsurg.2005.11.009. PMID: 16531147.\u003c/p\u003e\n\u003cp\u003e[10] Dunbar JP, Sulpice R, Dugon MM. The kiss of (cell) death: can venom-induced immune response contribute to dermal necrosis following arthropod envenomations? Clin Toxicol (Phila). 2019 Aug;57(8):677-685. doi: 10.1080/15563650.2019.1578367. Epub 2019 Feb 26. PMID: 30806093.\u003c/p\u003e\n\u003cp\u003e[11] Patel HJ, Patel BM. TNF-\u0026alpha; and cancer cachexia: Molecular insights and clinical implications. Life Sci. 2017 Feb 1;170:56-63. doi: 10.1016/j.lfs.2016.11.033. Epub 2016 Dec 3. PMID: 27919820.\u003c/p\u003e\n\u003cp\u003e[12] Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C, Norris A, Sanseau P, Cavalla D, Pirmohamed M. Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov. 2019 Jan;18(1):41-58. doi: 10.1038/nrd.2018.168. Epub 2018 Oct 12. PMID: 30310233.\u003c/p\u003e\n\u003cp\u003e[13] Parvathaneni V, Kulkarni NS, Muth A, Gupta V. Drug repurposing: a promising tool to accelerate the drug discovery process. Drug Discov Today. 2019 Oct;24(10):2076-2085. doi: 10.1016/j.drudis.2019.06.014. Epub 2019 Jun 22. PMID: 31238113.\u003c/p\u003e\n\u003cp\u003e[14] Roskoski R Jr. Properties of FDA-approved small molecule protein kinase inhibitors. Pharmacol Res. 2019 Jun;144:19-50. doi: 10.1016/j.phrs.2019.03.006. Epub 2019 Mar 13. PMID: 30877063.\u003c/p\u003e\n\u003cp\u003e[15] Sleire L, F\u0026oslash;rde HE, Netland IA, Leiss L, Skeie BS, Enger P\u0026Oslash;. Drug repurposing in cancer. Pharmacol Res. 2017 Oct;124:74-91. doi: 10.1016/j.phrs.2017.07.013. Epub 2017 Jul 13. PMID: 28712971.\u003c/p\u003e\n\u003cp\u003e[16] Conrad M, Angeli JP, Vandenabeele P, Stockwell BR. Regulated necrosis: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 2016 May;15(5):348-66. doi: 10.1038/nrd.2015.6. Epub 2016 Jan 18. PMID: 26775689; PMCID: PMC6531857.\u003c/p\u003e\n\u003cp\u003e[17] Wang S, Zhou D, Xu Z, Song J, Qian X, Lv X, Luan J. Anti-tumor Drug Targets Analysis: Current Insight and Future Prospect. Curr Drug Targets. 2019;20(11):1180-1202. doi: 10.2174/1389450120666190402145325. PMID: 30947670.\u003c/p\u003e\n\u003cp\u003e[18] Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000 Oct 13;103(2):239-52. doi: 10.1016/s0092-8674(00)00116-1. PMID: 11057897.\u003c/p\u003e\n\u003cp\u003e[19] Uitdehaag JC, Verkaar F, Alwan H, de Man J, Buijsman RC, Zaman GJ. A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets. Br J Pharmacol. 2012 Jun;166(3):858-76. doi: 10.1111/j.1476-5381.2012.01859.x. PMID: 22250956; PMCID: PMC3417414.\u003c/p\u003e\n\u003cp\u003e[20] Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002 Dec 6;298(5600):1911-2. doi: 10.1126/science.1072682. PMID: 12471242.\u003c/p\u003e\n\u003cp\u003e[21] Yao RQ, Ren C, Xia ZF, Yao YM. Organelle-specific autophagy in inflammatory diseases: a potential therapeutic target underlying the quality control of multiple organelles. Autophagy. 2021 Feb;17(2):385-401. doi: 10.1080/15548627.2020.1725377. Epub 2020 Feb 12. PMID: 32048886; PMCID: PMC8007140.\u003c/p\u003e\n\u003cp\u003e[22] Chen J, Ye C, Wan C, Li G, Peng L, Peng Y, Fang R. The Roles of c-Jun N-Terminal Kinase (JNK) in Infectious Diseases. Int J Mol Sci. 2021 Sep 6;22(17):9640. doi: 10.3390/ijms22179640. PMID: 34502556; PMCID: PMC8431791.\u003c/p\u003e\n\u003cp\u003e[23] Jia Y, Wang F, Guo Q, Li M, Wang L, Zhang Z, Jiang S, Jin H, Chen A, Tan S, Zhang F, Shao J, Zheng S. Curcumol induces RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling in hepatic stellate cells. Redox Biol. 2018 Oct;19:375-387. doi: 10.1016/j.redox.2018.09.007. Epub 2018 Sep 7. PMID: 30237126; PMCID: PMC6142373.\u003c/p\u003e\n\u003cp\u003e[24] Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ. 2005 Sep;29(3):151-9. doi: 10.1152/advan.00019.2005. PMID: 16109794.\u003c/p\u003e\n\u003cp\u003e[25] Yue Y, Zhang Q, Wu S, Wang S, Cui C, Yu M, Sun Z. Identification of key genes involved in JAK/STAT pathway in colorectal cancer. Mol Immunol. 2020 Dec;128:287-297. doi: 10.1016/j.molimm.2020.10.007. Epub 2020 Nov 25. PMID: 33248399.\u003c/p\u003e\n\u003cp\u003e[26] Hnasko TS, Hnasko RM. The Western Blot. Methods Mol Biol. 2015;1318:87-96. doi: 10.1007/978-1-4939-2742-5_9. PMID: 26160567.\u003c/p\u003e\n\u003cp\u003e[27] Hinderer EW 3rd, Moseley HNB. GOcats: A tool for categorizing Gene Ontology into subgraphs of user-defined concepts. PLoS One. 2020 Jun 11;15(6):e0233311. doi: 10.1371/journal.pone.0233311. PMID: 32525872; PMCID: PMC7289357.\u003c/p\u003e\n\u003cp\u003e[28] Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017 Jan 4;45(D1):D353-D361. doi: 10.1093/nar/gkw1092. Epub 2016 Nov 28. PMID: 27899662; PMCID: PMC5210567.\u003c/p\u003e\n\u003cp\u003e[29] Andrade C. The\u0026nbsp;P\u0026nbsp;Value and Statistical Significance: Misunderstandings, Explanations, Challenges, and Alternatives. Indian J Psychol Med. 2019 May-Jun;41(3):210-215. doi: 10.4103/IJPSYM.IJPSYM_193_19. PMID: 31142921; PMCID: PMC6532382.\u003c/p\u003e\n\u003cp\u003e[30] Lee EW, Seo J, Jeong M, Lee S, Song J. The roles of FADD in extrinsic apoptosis and necroptosis. BMB Rep. 2012 Sep;45(9):496-508. doi: 10.5483/bmbrep.2012.45.9.186. PMID: 23010170.\u003c/p\u003e\n\u003cp\u003e[31] Sosna J, Voigt S, Mathieu S, Lange A, Thon L, Davarnia P, Herdegen T, Linkermann A, Rittger A, Chan FK, Kabelitz D, Sch\u0026uuml;tze S, Adam D. TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death. Cell Mol Life Sci. 2014 Jan;71(2):331-48. doi: 10.1007/s00018-013-1381-6. Epub 2013 Jun 13. PMID: 23760205; PMCID: PMC3889832.\u003c/p\u003e\n\u003cp\u003e[32] Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. doi: 10.1038/nchembio711. Epub 2005 May 29. Erratum in: Nat Chem Biol. 2005 Sep;1(4):234. PMID: 16408008.\u003c/p\u003e\n\u003cp\u003e[33] Popa DS, Bigman G, Rusu ME. The Role of Vitamin K in Humans: Implication in Aging and Age-Associated Diseases. Antioxidants (Basel). 2021 Apr 6;10(4):566. doi: 10.3390/antiox10040566. PMID: 33917442; PMCID: PMC8067486.\u003c/p\u003e\n\u003cp\u003e[34] He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009 Jun 12;137(6):1100-11. doi: 10.1016/j.cell.2009.05.021. PMID: 19524512.\u003c/p\u003e\n\u003cp\u003e[35] Holbrook J, Lara-Reyna S, Jarosz-Griffiths H, McDermott M. Tumour necrosis factor signalling in health and disease. F1000Res. 2019 Jan 28;8:F1000 Faculty Rev-111. doi: 10.12688/f1000research.17023.1. PMID: 30755793; PMCID: PMC6352924.\u003c/p\u003e\n\u003cp\u003e[36] Sfikakis PP. The first decade of biologic TNF antagonists in clinical practice: lessons learned, unresolved issues and future directions. Curr Dir Autoimmun. 2010;11:180-210. doi: 10.1159/000289205. Epub 2010 Feb 18. PMID: 20173395.\u003c/p\u003e\n\u003cp\u003e[37] Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D'Orazi G. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging (Albany NY). 2016 Apr;8(4):603-19. doi: 10.18632/aging.100934. PMID: 27019364; PMCID: PMC4925817.\u003c/p\u003e\n\u003cp\u003e[38] Mohammad RM, Muqbil I, Lowe L, Yedjou C, Hsu HY, Lin LT, Siegelin MD, Fimognari C, Kumar NB, Dou QP, Yang H, Samadi AK, Russo GL, Spagnuolo C, Ray SK, Chakrabarti M, Morre JD, Coley HM, Honoki K, Fujii H, Georgakilas AG, Amedei A, Niccolai E, Amin A, Ashraf SS, Helferich WG, Yang X, Boosani CS, Guha G, Bhakta D, Ciriolo MR, Aquilano K, Chen S, Mohammed SI, Keith WN, Bilsland A, Halicka D, Nowsheen S, Azmi AS. Broad targeting of resistance to apoptosis in cancer. Semin Cancer Biol. 2015 Dec;35 Suppl(0):S78-S103. doi: 10.1016/j.semcancer.2015.03.001. Epub 2015 Apr 28. PMID: 25936818; PMCID: PMC4720504.\u003c/p\u003e\n\u003cp\u003e[39] Sleire L, F\u0026oslash;rde HE, Netland IA, Leiss L, Skeie BS, Enger P\u0026Oslash;. Drug repurposing in cancer. Pharmacol Res. 2017 Oct;124:74-91. doi: 10.1016/j.phrs.2017.07.013. Epub 2017 Jul 13. PMID: 28712971.\u003c/p\u003e\n\u003cp\u003e[40] Lamson DW, Plaza SM. The anticancer effects of vitamin K. Altern Med Rev. 2003 Aug;8(3):303-18. PMID: 12946240.\u003c/p\u003e\n\u003cp\u003e[41] Semkova S, Zhelev Z, Miller T, Sugaya K, Aoki I, Higashi T, Bakalova R. Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin. Anticancer Res. 2020 Apr;40(4):1963-1972. doi: 10.21873/anticanres.14151. PMID: 32234885.\u003c/p\u003e\n\u003cp\u003e[42] Calderon PB, Cadrobbi J, Marques C, Hong-Ngoc N, Jamison JM, Gilloteaux J, Summers JL, Taper HS. Potential therapeutic application of the association of vitamins C and K3 in cancer treatment. Curr Med Chem. 2002 Dec;9(24):2271-85. doi: 10.2174/0929867023368674. PMID: 12470246.\u003c/p\u003e\n\u003cp\u003e[43] Semkova S, Zhelev Z, Miller T, Sugaya K, Aoki I, Higashi T, Bakalova R. Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin. Anticancer Res. 2020 Apr;40(4):1963-1972. doi: 10.21873/anticanres.14151. PMID: 32234885.\u003c/p\u003e\n\u003cp\u003e[44] Kishore C, Sundaram S, Karunagaran D. Vitamin K3 (Menadione) suppresses epithelial-mesenchymal-transition and Wnt signaling pathway in human colorectal cancer cells. Chem Biol Interact. 2019 Aug 25;309:108725. doi: 10.1016/j.cbi.2019.108725. Epub 2019 Jun 22. PMID: 31238027.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colorectal cancer, Programmed cell death, Drug repurposing, Menadione, MAPK8","lastPublishedDoi":"10.21203/rs.3.rs-1406834/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1406834/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) is a conventional gastrointestinal malignant tumor, ranking third among all type of tumors and the fifth in clinical mortality. Programed cell death including apoptosis, autophagy and necroptosis that can be distinguished by their morphological and physiological differences. Although various researches and strategies were developed for anti-cancer drugs, there still lack of the effective therapies trigger cell programmed death to eliminate malignant colon tumor cells. In this study, we explored novel agents for inducing the programmed cell death through drug repurposing. We generated a high-throughput screening of a FDA Approved Drug Library, and identified Menadione, a synthetic analogue of vitamin K, as a promising candidate in regulating colon tumor programmed death. To further investigate the underlying mechanisms of Menadione in tumor programmed death, the selected gene of PCR array were performed and the data analysis showed significantly up-regulated gene by Menadione were ATG7 and MAPK8. The Go enrichment analysis also certify that MAPK8 may be an important regulation gene for Menadione induced cell apoptosis and necrosis in colorectal cancer. MAPK8 participated in multiple signaling pathways in GO enrichment analysis and KEGG pathway enrichment analysis, further demonstrated the key regulatory role of MAPK8. Thus, we identified Menadione may be a potential compound for MAPK8-targeting cascade. Owing to their FDA-approved status, Menadione might be used rapidly in the clinical treatment of tumor therapy. We demonstrated and provided new insights into the anti-cancer drug strategies.\u003c/p\u003e","manuscriptTitle":"Screening of a FDA-Approved Compound Library Identifies Menadione in Regulating Colon Cancer Programmed Death via MAPK8 Cascades","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-10 19:04:50","doi":"10.21203/rs.3.rs-1406834/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"acb9f13c-4ce4-4938-a6ba-b82eef928e3d","owner":[],"postedDate":"March 10th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-18T22:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-10 19:04:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1406834","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1406834","identity":"rs-1406834","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}