Lung cancer has a poor prognosis and a high mortality rate, and patients may develop multidrug resistance. Sparganii Rhizoma-Curcumae Rhizoma (HCSC), the classic herbal drug combination of traditional Chinese medicine (TCM), is commonly used in treating tumors, but its molecular mechanism is still unclear. in vitro the molecular mechanism of action of HCSC in treating lung cancer, as predicted by network pharmacology. Finally, we used the Symmap database to predict the relationship between the herb and TCM syndrome.  We explored the possible mechanisms underlying the antitumor effect of HCSC using network pharmacology. The bioactive components of HCSC and their targets were collected from the TCM Systems Pharmacology (TCMSP) database and PharmMapper. Gene Ontology (GO) and KEGG enrichment analyses were performed; the GeneMANIA platform was used for the functional enrichment analysis of the core targets and their neighboring genes. Molecular docking was performed between the bioactive components and core targets. HCSC freeze-dried powder was prepared, and the bioactive components were verified by liquid chromatography- (LC-) mass spectrometry (MS). Human lung adenocarcinoma H1975 cells were cultured to verify in vitro experiments on NCI-H1975 cells confirmed that HCSC can upregulate the mitochondrial-mediated caspase-dependent apoptosis signaling pathway by inducing the cleavage of caspase-3, caspase-9, and PARP, consistent with the network pharmacology prediction. Further, the qi deficiency and blood stasis associated with TCM syndrome can be treated with HCSC. A total of seven bioactive components were identified by network pharmacological analysis. Through enrichment analyses, it was found that the mechanism of action mainly involved mitochondrial-mediated caspase-dependent cell apoptosis signaling pathways. The results of molecular docking showed that the bioactive components in HCSC have a good affinity with the target proteins . LC-MS confirmed that formononetin and bisdemethoxycurcumin were present in the HCSC freeze-dried powder, consistent with the prediction. The results of  Lung cancer is one of the most common malignancies worldwide; it is associated with high mortality rates, and its incidence has been increasing gradually in recent years . HistopaThere has been no clear name for “lung cancer” in the history of TCM. Currently, it is mostly classified into the categories “feiji,” “xiben,” “lung distension,” or “hemoptysis.” Shen Jinao, a medical expert of the Qing Dynasty, wrote a book called “Incisive Light on the Source of Miscellaneous Disease,” which pointed out that “evil qi accumulates in the chest, blocks the airway, inhibits the functioning of qi, which causes phlegm-rheum, food accumulation, blood stasis, and the vacuity of right qi. Then it clumped.” Based on the basic theory of TCM and clinical experience, it has been believed that spleen qi is damaged after treatment. Radiotherapy, chemotherapy, and molecular-targeted drugs are heat toxins as per TCM and are more likely to wear qi and damage yin. Therefore, the deficiency of qi and yin was always reported throughout the entire process of lung cancer. This habitus makes patients more prone to qi stagnation and blood stasis.Sparganium stoloniferum. Curcumae Rhizoma, also known as turmeric, is a dried rhizome of common turmeric. According to TCM, the herbal couple Sparganii Rhizoma-Curcumae Rhizoma (HCSC) activates blood circulation to dissipate blood stasis. It is mentioned in “Records of Chinese Medicine with Reference to Western Medicine” that Zhang Xichun often used HCSC in clinical practice and called it “the most important medicine for blood quickening.” It was used to treat patients with lung cancer whose right qi could still fight against evil qi to prevent metastasis and recurrence after treatment. HCSC has been clinically proven to relieve the symptoms of patients with tumors, especially gynecological ones [Sparganii Rhizoma is the dried rhizome of cal ones . Modern cal ones –8. The acal ones . Curcumical ones –11.in vitro experiments. The detailed flowchart of the current study was shown in Network pharmacology is a discipline that combines systems biology and multidirectional pharmacology based on high-throughput omics data analysis, virtual computer calculation, and network database retrieval . Networkhttp://lsp.nwu.edu.cn/tcmsp.php) for Chinese herbal medicines; this platform captures the relationships between drugs, targets, and diseases [http://lilab.ecust.edu.cn/pharmmapper/) was used to simulate the molecular docking of the bioactive components [Homo sapiens,” was collected from the UniProt database (https://www.uniprot.org/) [The bioactive components of HCSC were retrieved from the TCM Systems Pharmacology (TCMSP) platform (diseases . The scrmponents . The ranot.org/) .http://ctdbase.org/) [https://auth.lifemapsc.com/) [https://www.disgenet.org/) [The Comparative Toxicogenomics Database (se.org/) , GeneCarsc.com/) , and Diset.org/)  were seahttps://string-db.org/) [A protein-protein interaction (PPI) network of candidate targets was constructed using the STRING database (db.org/) ; the “midb.org/)  to consthttp://www.genemania.org/) [P < 0.05). The ClueGO and CluePedia plugins in Cytoscape were used to perform the KEGG enrichment analysis of the core targets (P < 0.05); then, the pathway nodes in the enrichment network were manually selected. Candidate targets were converted to UniProtID using the UniProt software. The “search&Color Pathway” tool of the KEGG database (https://www.kegg.jp/kegg/pathway.html) [GeneMANIA (ia.org/)  was usedia.org/)  were useay.html)  was usedhttp://www.rcsb.org/) [G ≤ −5.0 kJ mol−1 was selected as the screening basis. The dominant conformation was analysed and plotted using Schrodinger.The HCSC bioactive components (ligands) were processed using ChemBio 3D software and stored in the mol2 format. Then, AutoDock Tools  were usesb.org/) . The oriHCSC consisted of 45 g of Sparganii Rhizoma and Curcumae Rhizoma each. All crude herbs were provided by the Zhejiang Provincial Hospital of Traditional Chinese Medicine. The herbs were mixed and boiled thrice in 1000 mL double-steamed water for 30 min each time, filtered by vacuum filtration, and concentrated on an induction cooker to about 100 mL. The liquid was then evaporated to dryness in a vacuum at −40°C. The final freeze-dried powder weighed 2.93 g; the yield was 3.25% . The freeze-dried powder of HCSC was then dissolved in double-steamed water (10 mg/mL) and filtered through a microporous filter for sterilisation.2 at 37°C. Cells in the logarithmic growth phase were used for all the experiments.Human lung adenocarcinoma H1975 cells  were kindly provided by Professor Zhou Caicun . The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained in a humidified chamber with 5% CO3 logarithmically growing H1975 cells/well were seeded in 96-well plates. After incubation for 24 h until the cell monolayer covered the bottom of the well, the cells were treated with varying concentrations of HCSC  for 24, 48, and 72 h. The 10% Cell Counting Kit-8  solution was added to each well, and the cells were cultured for another 1 h at 37°C. The optical density (OD) was measured at 450 nm using a multifunctional microplate reader . The inhibition rate is /average OD value of control group × 100%). The experiment was repeated three times in parallel. SPSS software was used to calculate the IC20 and IC50 values.A total of 6 × 105 cells/well) were seeded into 6-well plates. After incubation for 24 h, the cells were divided into the control (CG), low-dose (LD), and high-dose (HD) groups. The CG was treated without HCSC, while the LD and HD groups were treated with IC20 and IC50 proportions of HCSC for 24 h. The cells were collected and suspended in a 100 μl binding buffer containing 5 μl Annexin V-FITC and 10 μl PI. After incubation in the dark for 15 min at room temperature, 400 μl of binding buffer was added. Flow cytometry  was used to detect cell apoptosis in each group, and the FACSDiva Version 6.1.3 software was used for data analysis.Cells  = (1–0 h wound area/12 or 24 h wound area) × 100%. Image J was used to analyse the image data and determine the scratch area. The experiment was repeated three times in parallel.For the wound healing assay, cells were seeded in 6-well plates and incubated until 100% confluence. A sterile 200 5 cells/well) were seeded into 6-well plates. After incubation for 24 h, the cells were treated as described above for 24 h. The cells were scraped and collected. The protein of the H1975 cells was extracted with lysis buffer, and the concentration was measured. The sample volume was adjusted according to the internal reference protein to ensure the same amount of protein in each sample. The proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis based on their different molecular masses. Then, the proteins were transferred onto polyvinylidene difluoride membranes  using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The membrane was then immersed in 5% skim milk diluted with tris-buffered saline with 0.1% Tween-20 for 2 h at room temperature. After that, the membrane was cut into several bands and incubated with diluted antibodies (1 : 1000) at 4°C overnight. The antibody selected the relevant proteins in the KEGG enrichment pathway. The bands were washed with tris-buffered saline with 0.1% Tween-20 before incubation with HRP-conjugated secondary antibodies . At room temperature, a second incubation was required for 2 h. The bands were visualised using an electrochemiluminescence system . The experiment was repeated three times in parallel.Cells . The accurate molecular weight and secondary fragment information of the compound obtained from the total ion current diagram was compared with the information in the UNIFI TCM database to obtain the components.The filtered mother liquor of the freeze-dried HCSC powder was diluted to 1 mg/mL with double-distilled water, and the sample was injected. Mass spectrometry conditions were as follows: in the MShttps://www.symmap.org/) [SymMap (ap.org/)  containsA total of 111 bioactive components and 995 targets were identified in the TCMSP database. Eight potential bioactive components of HCSC and their corresponding targets were obtained . Ten targets were selected with the highest fit score in the PharmMapper database. A total of 113 targets were integrated and deleted after duplication . In the The above candidate targets were entered in the STRING database, and the “minimum required interaction score” was set at 0.4 to obtain a PPI network involving 106 potential targets. A “drugs-bioactive components-potential targets” network diagram was constructed using Cytoscape , 2. In aThe 18 core targets of HCSC were analysed using clusterProfiler  pV < 0..P < 0.05; 36 items in total). In the enrichment results, the P53 signaling pathway was selected and the candidate targets were mapped. This pathway has been reported in the literature to be closely related to lung cancer [The core targets were analysed using ClueGO and CluePedia for the KEGG pathway enrichment analysis  for 24, 48, and 72 h. HCSC can inhibit cell proliferation and exhibit a dose-time effect relationship. Compared with that in the control group, in the test group, the inhibition rate increased, and the increase was statistically significant. The ICter 24 h , 7.P < 0.01). Thus, HCSC can indeed induce apoptosis in H1975 cells, and apoptosis may be the main cause of death (The H1975 cells were treated with varying concentrations of HCSC (2.312–5.291 mg/mL) for 24 h. Compared with the control group, the rate of apoptosis in the test group increased significantly as the dose increased (15.17 ± 0.95 and 37.6 ± 1.64 vs. 6.33 ± 0.32) (of death .P < 0.01), and the mobility of the HD group decreased compared with that of the LD group (P < 0.05). After 24 h of scratching, the number of cells at the scratch site of the control group increased, and the scratch healing was obvious. The mobility of the LD and HD groups was reduced compared with that of the control group (P < 0.01); there was no significant difference in the mobility of the HD group compared with that of the LD group , caspase-3, caspase-9, and PARP . We found that compared with that in the control group, in the test groups, the expression level of caspase-3 in H1975 cells was significantly decreased (P < 0.05), whereas the expression level of cleaved caspase-3 was significantly increased (P < 0.05). Compared with that in the control group, in the test groups, the expression level of caspase-3 in H1975 cells in the HD group was significantly decreased (P < 0.01), whereas the expression levels of cleaved caspase-3, cleaved caspase-9, and cleaved PARP were significantly increased (P < 0.01). Compared with that in the LD group, in the test groups, the expression level of caspase-3 in the H1975 cells in the HD group was significantly decreased (P < 0.05), whereas the expression levels of cleaved caspase-3 and cleaved PARP were significantly increased (P < 0.01) and that of cleaved caspase-9 was significantly increased (P < 0.05)  .The total ion current diagram of the freeze-dried HCSC powder was obtained by LC-MS . Upon coSymMap provides herb-symptom relationships and symptom mechanism mapping. The results are as follows: HCSC contains 15 TCM symptoms, 11 TCM syndromes, 43 modern medicine symptoms, and 150 diseases. The TCM symptoms mainly include thoracic obstruction, Zhengjia, cardiodynia, abdominal mass, and amenorrhea, whereas the TCM syndromes involve phlegm-accumulation stasis, lung-spleen qi deficiency, and qi stagnation due to liver depression. The diseases mainly include SCLC, bladder cancer, T-cell immunodeficiency, pancreatic cancer, and other cancers, and the modern medicine symptoms include chest pain, dyspepsia, neoplastic growth, angina pectoris, and menopause.TCM is usually applied in combination; it is a therapeutic treatment mode comprising multiple pharmacological bioactive components that cooperate with different pathways. The mechanism of HCSC with multiple components, multiple targets, and multiple pathways has limited in-depth exploration. However, the emergence of network pharmacology has solved this problem.In this study, we collected seven potential bioactive components of HCSC, among which hederagenin is a common component of Sparganii Rhizoma and Curcumae Rhizoma. Moreover, sitosterol , formonoIn vitro experiments in H1975 cells were used to clarify the activity of HCSC in treating lung cancer and to further verify the relevant molecular mechanisms predicted by network pharmacology. Experimental results showed that HCSC can significantly inhibit the proliferation of H1975 cells, promote apoptosis, and reduce cell migration. HCSC plays an all-around role in the treatment of lung cancer and is expected to overcome the resistance of EGFR-TKI caused by the T790M mutation. Western blotting results showed that the protein expression levels of cleaved caspase-3, cleaved caspase-9, and cleaved PARP in H1975 cells after HCSC treatment were significantly higher than the levels of those in the control group. This suggests that HCSC can upregulate the mitochondrial-mediated caspase-dependent apoptosis signaling pathway by inducing the cleavage of caspase-3, caspase-9, and PARP, consistent with the aforementioned network pharmacology prediction pathway. The activity of HCSC in the treatment of lung cancer is dose- and time-dependent to a certain extent. Because there were fewer concentration gradients in our experiment, more groups still need to be set to verify the above conclusions. LC-MS confirmed that formononetin and bisdemethoxycurcumin were present in the freeze-dried powder of HCSC, which matched the prediction in the database. This suggests that the in vitro antitumor effect of the freeze-dried powder of HCSC may be dominated by formononetin and bisdemethoxycurcumin.This study also explains the relationship between disease-syndrome-herb through the analysis of the SymMap database. According to the basic theory of TCM, lung cancer was historically classified into the Zhengjia category. At present, there is no unified standard for the classification of TCM syndromes in lung cancer. The deficiency of lung-spleen qi and the stagnation of qi and blood stasis are common TCM syndromes in patients with advanced lung cancer, and these syndromes often manifest as dyspepsia and chest pain , 37. Thiin vitro experiments to reach the following conclusions: (1) the core bioactive components of HCSC were sitosterol, formononetin, and bisdemethoxycurcumin; (2) HCSC can upregulate the mitochondrial-mediated caspase-dependent apoptosis signaling pathway by inducing the cleavage of caspase-3, caspase-9, and PARP; (3) the qi deficiency and blood stasis syndrome would be the most suitable for treatment with the HCSC. Thus, this study enhanced the therapeutic potential of HCSC in lung cancer.This research innovatively used network pharmacology and


Stevia rebaudiana is a South American plant, the cultivation of which is increasing worldwide due to its high content of sweet compounds. Stevia sweetness is mainly due to steviol glycosides, that are ~250-300 times sweeter than sucrose. Many studies have suggested the benefits of Stevia extract over sugar and artificial sweeteners, but it is still not a very popular sugar substitute. This review summarizes current data on the biological activities of S. rebaudiana extract and its individual glycosides, including anti-hypertensive, anti-obesity, anti-diabetic, antioxidant, anti-cancer, anti-inflammatory, and antimicrobial effects and improvement of kidney function. Possible side effects and toxicity of Stevia extract are also discussed. CFTR, cystic fibrosis transmembrane conductance regulator; GFR, glomerular filtration rate; LPS, lipopolysaccharide; MDCK cells, Madina-Darby canine kidney cells; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear erythroid factor 2; TRPM5, transient receptor potential cation channel subfamily melastatin member 5; TPA, 12-O-tetradecanoyl-phorbol-13-acetate.Stevia of the Asteraceae family includes 230 species, but only one of them, Stevia rebaudiana Bertoni produces sweet steviol glycosides. It was earlier thought that S. phlebophylla also has this property, but new research denies this .S. rebaudiana is a perennial shrub growing in South America, in particular in Brazil and Paraguay, where it is also known as “Honey Leaf”, “Sweet-Leaf” or “Sweet-Herb”. Stevia preparations are used in many forms such as fresh and dried Stevia leaves, Stevia leaf powder, extracts and liquid concentrates. Stevia extract is a great alternative for synthetic sweeteners being approximately 200 to 300 times sweeter than sugar . Many sal., 2017). Safetyal., 2017). Stevia preparations are steviol diterpene glycosides .Stevia has been known to people from ancient times. The Guarani Indians called Stevia “Ka-a He-e”, which means “sweet grass”, and used it to savor bitter drinks such as mate. There are some reports that Stevia was already known in Spain in the 16th century. However, other Europeans learned about the plant only in the late 19th century, after Stevia was introduced and promoted by the botanist Moises Santiago Bertoni . Bertonal., 2008). In 190al., 2008). Duringal., 2015).Stevia was introduced in Japan and research was started to evaluate its beneficial potential for human health. Since then, the Japanese actively use this sweetener in a variety of foods and Japan is one of the major producers of Stevia now . In 201den, 2013). In addank, 2002). S. rebaudiana contain more than 30 different steviol glycosides, among which stevioside and rebaudioside A are present in the highest levels . Rebaudcio, 2015). The cocio, 2015; Goyal ecio, 2015).Stevia leaves are glycosides, stevioside, several types of rebaudiosides (from A to F), steviolmonoside, rubusoside, dulcoside A, and steviolbioside . Less cal., 2002). Rebaudrth, 2020). Despitrth, 2020). Rebaudrth, 2020).Stevia, namely rebaudioside U, T, R and S . Stevioal., 2016). Most oal., 2016; Purkayaal., 2016). Sweet cio, 2015). ProcesStevia leaves contain proteins, carbohydrates, lipids, dietary fibers, oils, vitamins, and phenolic compounds. Dried Stevia leaves contain (per 100 g of dried mass) 11.2-16.0 g proteins, 61.9 g carbohydrates, 1.9-3.73 g lipids and 6.8-15.2 g dietary fiber .Stevia differs in dry and fresh leaves and depends on the method of processing or extraction . Stevia leaves contain a number of phenolic compounds that exhibit strong antioxidant properties. The content of total polyphenols and flavonoids in methanolic extracts was estimated as 25.18 mg/g and 21.73 mg/g, respectively , vitamin B2 (0.43 mg/100 g), folic acid (52.18 mg/100 g) . This hal., 2011). MoreovStevia contains stevial glycosides. Rubus suavissimus also known as sweet tea, contains steviol monoside, rebaudioside A, B, C, D, F, M, stevioside, steviolbioside, and rubusoside . In addal., 1992). Stevia have been studied for over 100 years. Both earlier and current studies not only confirm the safety of Stevia leaf preparations but also find more and more benefits of its consumption for human health. Next, we will summarize current data on the established and potential pharmacological effects of Stevia extracts and its individual components in various medical models .2O2 exposure compared with the control cell group . These al., 2017). One stal., 2012). Anotheal., 2007).Stevia glycosides were found to reduce lipoperoxidation and decrease hydroperoxide and protein carbonyl content in Cyprinus carpio . Stevioal., 2017). In addStevia extracts depend on the methods of extract processing including stages of drying and extraction. Thus, the highest total content of phenols was observed in glycol-aqueous extracts from Stevia leaves, with lower phenol content in aqueous extracts and the lowest levels in ethanol extracts  showed that the best method of drying to keep high antioxidant potential was hot air drying at 180 °C. Under this regime, the content of antioxidants was 2-3 times higher than when using other drying methods . Comparal., 2015).Cancer is the second leading cause of mortality in the world. The most common forms of cancers in 2017 were breast cancer, colorectal cancer, prostate cancer, and lung cancer. Therefore, the search for agents that may help in cancer treatment or prevention is extremely important .Stevia components in chemotherapy of cancer . High cal., 2005) .Stevia leaves significantly inhibited the NF-κB-mediated gene expression that was stimulated by bacterial lipopolysaccharide (LPS). Such inhibition of NF-κB signaling was closely associated with a decrease in levels of interleukin-6 and monocyte chemoattractant protein-1. It was suggested that Stevia could be used for treatment of immune diseases such as rheumatoid arthritis and lupus . Stevioal., 2010). Stevioal., 2010). Austroal., 2013). The meal., 2011; Arya etStevia also has antibacterial effects in the oral cavity . Dentalras, 2013).Stevia extracts against microorganisms that cause tooth decay. Extracts were obtained from dried S. rebaudiana leaves using hexane, methanol, ethanol, ethyl acetate or chloroform as solvents. The antimicrobial activity of these five extracts against 16 bacterial strains of the genera Streptococcus and Lactobacillus was evaluated by the well-known diffusion method. For the four Lactobacillus species, the inhibition zones obtained between 12.3 and 17.3 mm were somewhat larger with ethyl acetate and chloroform extracts, suggesting that these bacteria were the most susceptible microorganisms. In another study, four groups of rats were fed with stevioside, rebaudioside A or sucrose, which were added to the main diet. There were significant differences in the rates of sulcal caries and Streptococcus sobrinus count between a group fed diet with 30 % sucrose and the other 3 groups. There were no differences between groups fed stevioside or rebaudioside A and groups without additives. Thus, it can be argued that neither stevioside nor rebaudioside A are cariogenic  also stal., 1992). An in zky, 2010). Cariesal., 2013). Due tosma, 2015). Moreoval., 2017).Diabetes is a group of diseases in which insulin is either not produced in sufficient quantities (type 1 diabetes) or cannot be used effectively due to insulin resistance (type 2 diabetes). As of 2019, there were 464 million people with diabetes in the world; for comparison, in 2000, 175.4 million of patients with diabetes was recorded. According to the International Diabetes Federation, by 2040 this number will increase to 642 million . About Stevia has been used not only as a sweetener but also as a medication in the treatment of diabetes and hyperglycemia in traditional medicine in Brazil and Paraguay . The an Figure 4. One stumad, 2018). An incmad, 2018). In addmad, 2018). Glycosmad, 2018). Consummad, 2018). A numbal., 2010; Mehta eal., 2010; Singh aal., 2010; He et aal., 2010). Steviaal., 2017). Moreoval., 2018). The abal., 2018). Groupsal., 2017). Furtheal., 2015). Howeveal., 2008; Ajami eetc. . S. rebaudiana extracts were administered to rats for 20, 40, and 60 days and results showed that rats treated with Stevia extract for 20 days did not significantly differ from the control group in the arterial pressure . The hypotensive effect on both systolic and diastolic blood pressure was dose-dependent for intravenous doses of 50, 100 and 200 mg/kg in conscious SHR. The hypotensive effect lasted for more than 60 min with a dose of 200 mg/kg. Thus, stevioside was effective in blood pressure reduction . In anolis, 1995). Howevelis, 1995). In onelis, 1995). Consumlis, 1995). Rebaudlis, 1995). Systollis, 1995).S. rebaudiana support its anti-obesity properties . Abdomi Figure 4. In one Cha, 2010). In addCha, 2010). Stevia extract was only due to the difference in calorie intake; therefore, consumption of Stevia extract did not affect the feeling of hunger and appetite . It alsStevia extract could prevent the adverse effects of high fat, high sucrose diet on lipid profiles, total antioxidant capacity and histopathologic factors in obese rats . In addal., 2020).The kidneys are one of the most important organs, their main function being to excrete end- and side-products of metabolism. The kidneys are also involved in regulation of blood pressure, water-salt balance and red blood cell formation . DiabetStevia leaves caused systemic and renal vasodilation, hypotension, diuresis and natriuresis . The filis, 1995). Yuajitlis, 1995) investial., 2018). Stevioal., 2018).Stevia dry extract defined by the Scientific Committee on Food of the European Food Safety Authority and Food and Drug Administration is 4 mg/kg body mass . One anal., 2015), but thStevia extract reduced the fertility of rats by up to 21 % compared with control group of rats. Fertility remained reduced by 47 % even after a 50-60 day recovery period . In SteStevia safety is that for over 1500 years of continuous use by Paraguayans, there have been any reports of adverse effects. Additional confirmation of the safety of Stevia consumption is the absence of reports on side effects of any kind in Japanese populations where Stevia has been consumed in large quantities in recent years . The maRao, 2005). In addal., 2010; Nikiforal., 2010; Uçar etal., 2010). Howeveal., 2016).S. rebaudiana, a perennial plant native to Paraguay, has come to be cultivated around the world as a source of high-potency sweetener with no caloric value. Two main steviol diterpene glycosides, stevioside and rebaudioside A, that are present in high levels in Stevia leaves provide the sweet taste of the plant and are 150-450 times sweeter than sucrose to human taste buds. Pure sweet glycosides and crude Stevia extracts with 50 % of glycosides are actively used in the food market. A number of preclinical and clinical studies suggest potential therapeutic and pharmacological applications for Stevia extracts and their individual compounds because they demonstrate no toxicity in experimental trails and exhibit health-promoting activities. In addition to different glycosides, Stevia leaves contain many other compounds like flavonoids and fatty acids that together provide the diverse biological properties of the plant. Thanks to these components, Stevia products stimulate insulin production in diabetics, improve polycystic kidney disease, have chemotherapeutic action in cancer and possess powerful antibacterial, antioxidant and immunomodulating properties  and a Discovery grant from the Natural Sciences and Engineering Research Council of Canada (#6793).The authors declare that they have no conflict of interest.Victoria Peteliuk and Lesia Rybchuk collected literature and wrote the original draft, prepared the figures and tables; Maria Bayliak performed analysis, review and edited the manuscript, helped to prepare and arrange tables and figures; Kenneth Storey performed review, editing and provided valuable discussion; Oleh Lushchak: provided funding acquisition, idea and design of the article, supervising, review, and editing. All authors read and approved the final manuscript.


Evidence is limited on how to synthesize and incorporate the views of stakeholders into a multisite pragmatic trial and how much academic teams change study design and protocol in response to stakeholder input. This qualitative study describes how stakeholders contributed to the design, conduct, and dissemination of findings of a multisite pragmatic clinical trial, the COMprehensive Post-Acute Stroke Services (COMPASS) Study. We engaged stakeholders as integral research partners by embedding them in study committees and community resource networks that supported local sites. Data stemmed from formal focus groups and continuous participation in working groups. Guided by Grounded Theory, we extracted themes from focus group and meeting notes. These were discussed as a team and with other stakeholder groups for feasibility. A consensus approach was used. Stakeholder input changed many aspects of the study including: the care model that treated stroke as a chronic condition after hospital discharge, training for hospital-based providers who often lacked awareness of the barriers to recovery that patients face, support for caregivers who were essential for stroke patients’ recovery, and for community-based health and social service providers whose services can support recovery yet often go underutilized. Stakeholders brought value to both pragmatic research and health service delivery. Future studies should test the impact of elements of study implementation informed by stakeholders vs those that are not. A primary goal of the Patient-Centered Outcomes Research Institute (PCORI) is to advance the science of community engagement in research . To dateClinicians, social scientists, and funders have demonstrated increased interest in how to effectively involve stakeholders in all phases of research so that research is relevant, meaningful, and actionable to those receiving the intervention, delivering the intervention, and translating the model into standard practice and policy. Initially, much of the research focused on patient and public involvement in the early stages , including projects focused broadly on the patient experience, or narrowly on specific population groups or specific areas of research –13. MoreThis paper describes how stakeholders contributed to the design, conduct, and dissemination of findings of a multicenter pragmatic clinical trial and the changes the academic team made to the study in response to their input. The cluster-randomized COMprehensive Post-Acute Stroke Services (COMPASS) Study investigated the effectiveness of implementing an evidence-based, comprehensive, post-acute stroke transitional care model compared with hospitals’ usual care. The study design and methods – including methods used to engage stakeholders – are published –23. ThisThe COMPASS Study was a pragmatic cluster-randomized controlled trial conducted in 40 hospitals with approximately 10,000 stroke and transient ischemic attack (TIA) patients in North Carolina. Intervention hospitals employed a novel transitional care model, as well as an additional set of billing codes to provide financial incentives, in order to change provider behavior and increase quality of care and recovery outcomes for stroke and TIA patients. The study provided small supplemental funding to hospitals to offset research-related costs only. The primary trial results are published .The COMPASS model was initially developed in collaboration with the Wake Forest Baptist Health Comprehensive Stroke Center clinical team. It incorporated their prior experience with stroke transitional care through the TRAnsition Coaching for Stroke (TRACS) Program and evidence from the most current scientific literature .. Our primary goal for stakeholder engagement in the COMPASS Study was to be responsive to both patient and caregiver needs, to treat recovery from stroke as a chronic condition, to create a realistic workflow for hospital-based providers, and to link patients to community-based services to address social determinants of health to maximize recovery.Details on the COMPASS Study’s non-traditional research partners and the rationale for their inclusion have been published . BrieflyFig. . Our prWe used qualitative methods to investigate stakeholder needs and priorities, which enabled us to more fully capture insights and reactions than would be possible with quantitative methodologies with pre-defined response options. Qualitative methods also allow new areas of inquiry to emerge and can reveal perspectives that researchers may not be able to foresee . TransitEliciting and incorporating stakeholder perspectives involved embedding stakeholders into study committees, assembling resource networks, conducting focus groups, and group discussions. These group interactions encouraged stakeholders to talk to one another, ask questions, and comment on one another’s experiences and perspectives, leading to more nuanced recommendations. Furthermore, the iterative process of holding multiple discussions over time with diverse groups of stakeholders helped refine recommendations and confirm that recommendations correctly reflected their ideas and were feasible with existing resources.We collected input from stakeholders using committees and resource networks, as described below.At the state level, we formed a large group of partners to be both inclusive and to leverage the tremendous expertise across the state. We purposefully included diverse perspectives, including urban/rural lived experiences of patients and providers, as well as patients and caregivers who represented varied income and educational levels. Some stakeholders on this committee were embedded in the study’s Steering Committee and subcommittees, which met weekly, so that they could have continuous input throughout the study. Some stakeholders were consulted outside the regular meeting schedule, most frequently by the study’s Principal Investigator (PI) and the Director of Implementation, both clinicians with extensive familiarity with North Carolina’s stroke system of care. Other stakeholders provided important insights via focus groups and interviews. See Supplement 1 for an example of what one focus group of elder, rural residents revealed to researchers, how that information was used, and how these stakeholders were informed that we had made changes to the study in response to their feedback.At the local level, we guided the post-acute care coordinator at each of the 40 participating hospitals to develop a COMPASS Community Resource Network (CRN). These networks of community-based health and social service providers helped the post-acute care coordinator link patients to resources outside the hospital that could support recovery. The goal was to incorporate local knowledge into the intervention to maximize successful implementation. Each CRN included a representative from aging services, a pharmacist, a home health provider, and a rehabilitation provider. Some CRNs also included a representative from the local health department, a community paramedic, a faith leader, a local stroke survivor, a local caregiver, a transportation service representative, and/or a social worker, reflecting each community’s unique resources and strengths. As part of implementation, each CRN engaged with the study’s Director of Implementation and other implementation and training team members during on-site day-long hospital meetings at the beginning of the study. CRNs also provided feedback to the study team by participating in bi-weekly group conference calls for problem solving. CRNs also participated in two surveys in which they shared challenges they had experienced as well as solutions. These data collection activities allowed the study team to continuously learn from those delivering the intervention.All stakeholder engagement activities were submitted to the Wake Forest University Health Sciences Institutional Review Board (IRB). The IRB classified (and approved) some stakeholder engagement activities as “Human Subjects Research” and other stakeholder engagement activities as “Not Human Subjects Research.” For IRB-approved research activities , participants provided verbal consent. For activities that did not meet the IRB definition for research , there was no consent process.et al. 2020 for description of tool)  COMPASS has really done for us is identify some holes that are in our program and the speech therapy is a prime example… I didn’t know before starting COMPASS that these things were getting missed or they weren’t being done or the patients were struggling like they were…. You don’t know what you don’t know, and I certainly had no idea. So we have been able to work through and do some education and set up some processes where we can limit these missed therapies and missed opportunities for incorporating resources for the patient.”Importantly, by bringing together patients, community-based service providers and hospital-based providers, it became clear that there were gaps in care that patients could easily identify and that providers were unaware of but eager to address. As one stroke coordinator shared with us,The cross-sector collaboration in the study also gave community agencies an entry point into their local hospitals which, in the past, they had not been able to establish on their own. There were multiple points throughout the study in which these stakeholders drove engagement activities that clearly strengthened the study. For example, the study pharmacist saw opportunities, knew of an existing network of community-based pharmacies that offered enhanced services, and had critical personal connections that made it possible for the study team to link a community-based specialty pharmacist to each of the 40 hospitals (via their CRNs). These community pharmacies often were able to provide to patients evidence-based interventions . The study team recognized the value that these community pharmacists offered, but, over the course of implementation, they emerged as markedly more critical partners in care than the team had initially expected. If a pharmacist had not been on the study team, the team would not have been aware of the opportunities he identified; nor would the team have had the credibility or social capital that he had in his professional network to forge these statewide partnerships and connect patients with community services to support their recovery. Expanding beyond the hospital perspective enabled us to treat the patient holistically.Not knowing who to call as soon as they recognized they had cognitive and/or physical deficits that were not detected in the hospital.Survival and full recovery require that the patient has an able and willing caregiver, yet many stroke survivors live alone or have ailing spouses who themselves need caretaking.Not having a roadmap to follow to recovery.The study team made several observations after including diverse stakeholders in relation to socioeconomic status, urban/rural location, and racial representation. First, regardless of socioeconomic status, location, or race, patients and caregivers voiced the same problems with usual care after stroke:also assesses the caregiver’s ability to care for the patient. The intervention also calls for a single person, trained in stroke, to be the clear contact person for the patient; and this information is communicated to the patient and/or family in a myriad of ways . The guidance stakeholders gave for a roadmap to recovery (Care Plan) is described in Table These unmet needs were universal and shaped the intervention. It was particularly eye opening to hear from highly resourced patients and caregivers that they – in spite of their social and economic advantages – were overwhelmed by having to managing post-acute care. They told us that they did not see how people with fewer resources could cope or ever fully recover. Their lived experience pushed the team to design an intervention that assesses the patient comprehensively, and Second, stakeholder diversity in terms of rural/urban and racial representation produced creative solutions to common problems. Rural hospitals had clinical teams that were exceptionally effective at anticipating and successfully addressing implementation problems. They attributed this prowess to knowing most people in the small hospital and community and being able to leverage those relationships. The solutions developed at rural sites were shared with and then adopted by urban sites. For example, a rural site could not identify the protocol-specified RN within the hospital system to serve as the main contact to the patient. But a clinician did know of a community paramedic program, rooted in the African-American community that was already making home visits. The site argued that a community paramedic, who was trusted by community members, could effectively function in the protocol-specified role and the protocol was amended. This solution was then used in other sites.Several clinical stakeholders who were African-American and started as advocates for their African-American patients became trail blazers in implementing adaptations of the intervention after the measurement period (during the sustainability phase) to further address their needs. For example, to address remaining transportation barriers, they consolidated members of the care team to one location, and made the comprehensive assessment of the patient a televisit. The study team’s next study is to test the effectiveness and implementation of a televisit vs in-person visit for comprehensive post-acute stroke care now that Medicare is covering telemedicine during the COVID pandemic and this form of the intervention is sustainable.In this study, we actively elicited and incorporated input from a diverse set of stakeholders in one of the first large-scale pragmatic clinical trials of transitional care in the USA. This qualitative assessment highlighted ways that patients and caregivers shaped the design of the intervention, enhanced caregiver and clinician training, designed ways to improve patient recruitment and participation, and made patient-facing materials more useful to other stroke patients and their caregivers. Furthermore, it revealed ways that engagement of acute care hospitals’ providers and administrators, post-acute care providers, local pharmacists, and primary care physicians facilitated uptake of a complex intervention. Unexpectedly, it also revealed ways that stakeholders themselves benefited from engagement with the research team.The majority of the literature to date on stakeholder engagement in research has provided general guidelines or has focused on the barriers to successfully engaging stakeholders . A commoPatient and family stakeholders were typically delighted when asked to join or advise the study team. Indeed, many patients sought out the study team when they heard about it through the local media. Patients and caregivers were typically motivated to collaborate as partners when learning that the research team wanted to learn from their experience to improve care for others. Community-based health and social service providers were similarly motivated to serve as stakeholders, even those who had no prior research experience, due to the shared mission of improving health and wellbeing of either stroke patients in particular or older individuals more broadly. Patients were open and honest with their input and feedback. At times, patients and families would couch their critiques of the hospital care they received by explicitly praising their providers . Patient and family stakeholders frequently expressed that being able to contribute to the study was critical to their own healing. Specifically, they stated that bringing awareness to gaps in care and being part of the change that they hoped would reach thousands of other patients helped them rebuild their confidence in public speaking, redirect their grief or anger into something productive, make sense of their new life, and fulfill their new purpose in life.This paper has limitations. Due to resource constraints it was simply not feasible for the study team to deeply engage with all stakeholders and also transcribe and tag all data with codes. Engagement activities were tracked but some were captured in less detail, truncated or summarized in REDCap forms. However, in those cases we often uploaded documents that captured rich details about the engagement activities. The strength of this paper is that the team documented the vast majority of engagement activities and systematically engaged stakeholders from design to dissemination and acted on stakeholder advice.https://www.nccompass-study.org/), to education of hospital-based clinicians on what it means to deliver patient-centered care, to strengthened community partnerships, to sharing evidence that informed policymakers about the need for primary and secondary prevention care in stroke. Very importantly, stakeholder involvement in the study produced a new patient-centered consent model for future pragmatic trials [Stakeholder involvement in the COMPASS Study produced lasting benefits to stakeholder groups beyond the scope of the study, from publicly available, patient-friendly educational materials . She changed the language used on the consent form and study brochures because she did not want a single patient to think they would be receiving suboptimal care if assigned to the control group (for fear that perception would undermine their recovery). While the study team could offer language the IRB approved, she could offer language that would not turn patients away from the study.A common charge against stakeholder involvement is the amount of time and resources required. We agree that, for stakeholder engagement to be meaningful, it does require paid research effort, study infrastructure, and payment for the time that patients and community organizations provide to the study. The study budget was established in 2015. It was important to reimburse stakeholders equitably and consistently. See Supplement 4 for our policy and procedures for reimbursing stakeholders. The study paid $20 per hour to patient and caregiver stakeholders for their time spent preparing for meetings, attending meetings, reviewing materials, and travel when necessary, etc. For health professional and corporate stakeholders there was overlap between their established (paid) work and study needs, therefore they were not compensated at an hourly rate. All stakeholders were eligible for compensation for their time in focus groups and interviews which the IRB defined as research. It was necessary to balance compensation across a large stakeholder base while not limiting input from stakeholders or exceeding study budget. It is now 2020, and as our team prepares other grants and associated budgets we have increased the hourly rate to 30–40 dollars per hour (depending upon the size of the grant) as we feel that 20 dollars per hour should be increased for the value that stakeholders bring to each study.It should be noted that a collaborating advocacy organization declined reimbursement. A community-based organization negotiated a 10,000 dollar per year flat rate. Reimbursement went to the organization, not to the individuals engaged in the study. Unlike community-based participatory research, stakeholder engagement in research can lie along a continuum of meaningful participation, which provides some flexibility in time and resource investment. We believe that the short-term costs of investing in stakeholder engagement pays off over the long term. For instance, it may take longer to create a consent process and consent form that patients, caregivers, and the IRB approve, but it may support patient recruitment and hospital retention over the course of the study.Future studies should directly test the impact of elements of study design, implementation, and dissemination that were informed by stakeholders vs those that were not to continue to advance the science of engagement in clinical research. Future research should also quantitatively test the effect of stakeholder engagement on patient recruitment, patient or provider behavior, and patient-centered outcomes. Another area ripe for study is understanding how patients personally benefits by contributing to research projects  and if and how that might affect their own recovery. This will require additional time and resources but is critical to strengthening the inclusion of stakeholders in the design and implementation of future studies and programs.


Protein degradation by the Ubiquitin-Proteasome System is one of the main mechanisms of the regulation of cellular proteostasis, and the E3 ligases are the key effectors for the protein recognition and degradation. Many E3 ligases have key roles in cell cycle regulation, acting as checkpoints and checkpoint regulators. One of the many important proteins involved in the regulation of the cell cycle are the members of the Histone Deacetylase (HDAC) family. The importance of zinc dependent HDACs in the regulation of chromatin packing and, therefore, gene expression, has made them targets for the design and synthesis of HDAC inhibitors. However, achieving potency and selectivity has proven to be a challenge due to the homology between the zinc dependent HDACs. PROteolysis TArgeting Chimaera (PROTAC) design has been demonstrated to be a useful strategy to inhibit and selectively degrade protein targets. In this review, we attempt to summarize the E3 ligases that naturally ubiquitinate HDACs, analyze their structure, and list the known ligands that can bind to these E3 ligases and be used for PROTAC design, as well as the already described HDAC-targeted PROTACs. Proteins inside the cells are subjected to a well meshed quality control system that is comprised of the Ubiquitin-Proteasome System (UPS), autophagic lysosomes, and the endoplasmic reticulum. Misfolded proteins will reach the endoplasmic reticulum where a refolding process will be attempted by the Heat-Shock Proteins (HSPs). Should this process fail, the misfolded proteins will be transported back to the cytoplasm where they will be finally degraded by either the UPS or lysosomes . AutophaC-terminal end of Ub (The UPS system is comprised of the 26S proteasome and an ensemble of three enzymes that carry out substrate ubiquitination in cascade: the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin ligase (E3) [nd of Ub  7]..C-terminUbiquitination in the substrate protein may happen by either the addition of one Ub molecule on one lysine residue (monoubiquitination) or several lysine residues (multiubiquitination); or the addition of several Ub molecules on one single lysine residue (poliubiquitination) . Poli-UbThe type of E3 ligase will determine what protein is going to be ubiquitinated and which is the E2 ligase that is going to take part in the process, as the E3 ligase has specific recognition domains for both proteins . There a2+ are coordinated to cysteine residues in the RING finger motif that are recognizable by the following sequence: Cys-X2-Cys-X(9−39)-Cys-X(1−3)-His-X(2−3)-Cys-X2-Cys-X(4−48)-Cys-X2-Cys .,84.N-terCRBN system can adopt different conformations. The conformational change undergone by CRBN is reminiscent of the opening and closing movement of the thumb: closed when it is near the hand and opened when it is away from the hand. When CRBN interacts with Ikaros, the conformation is opened (PDB code 6H0F), but when it interacts with CK1 the conformation is closed (PDB code 5FQD)   71,84].,84.CRBN C-terminal end and the SPOP protein at the N-terminal end. The SPOP protein, whose name stands for speckle-type POZ proteins due to the dotted pattern it produced in the nucleus of the cells, is the substrate-recognition protein of this Cullin-RING E3 ligase complex that also belongs to the BTB  family (The Cullin-3 protein that works as the scaffold of this system interacts with Rbx1 at the ) family  85,86].,86.C-terDue to the nuclear residence of SPOP, it recruits as substrates different nuclear proteins that are at the end of many cellular pathways. Works carried out by Cuneo M. J. et al. listed a total of 26 SPOP substrates that ranged from nuclear receptors, epigenetic modifiers, transcription factors, and cell cycle modulators, to nuclear phosphatases . SubstraN-terminal end; the BTB domain in the center part of the protein; and a C-terminal BTB and C-terminal Kelch (BACK) domain  domain at the ) domain . This lain  has been shown to regulate the entrance into the metaphase of cell division as it delays chromosome condensation and is considered to be a tumor suppressor ,93. CHFRN-terminal end, the RING domain, and the CRD (Cystein-Rich Domain) at the C-terminal end (CHFR is made up of three domains: FHA (ForkHead-Associated) domain at the inal end a.2+ ions located in four Zn-binding domains, of which the last conforms the PAR-Binding Zinc finger (PBZ). This PBZ is the binding site for PolyADP-ribose (PAR) that constitutes a post-translational modification that rises from DNA damage. Despite not being crystalized in complex with CHFR, Oberoi J. et al. were able to crystalize the CRD of CHFR in complex with PAR-structurally related ligands . . N-termi2+ ions coordinated with cysteine and histidine residues: six located in the N-terminal domain, two in the RING domain and one in the C-terminal domain. The N-terminal domain is formed by the N and C lobe perpendicular to one another, that are made up of small antiparallel β-sheets and a α-helix. This N-terminal domain is able to weakly interact with the DNA-binding domain (DBD) of p53, thus contributing to the ubiquitination of this tumor suppressor. On the other side, the C-terminal domain presents a disordered N-terminal end, whereas the C-terminal end presents a small globular structure near the last zinc ion. Additionally, residues Ala249 and Ile256 create an interacting surface with the p53 tetramerization domain (TET) . T. TN-termecessary . Oligometructure  129]. I. IN-term of MDM2 .N-terminal end of these ligases and is comprised of three hydrophobic pockets that interact with residues Phe19, Trp23, and Leu26 which are part of the α-helix of p53 that interacts with MDM2 and MDMX [The p53 binding site is located at the and MDMX . The intand MDMX . The p53and MDMX ,131. Due to the implication of MDM2 in cancer regulation, there is a great interest in the development of selective inhibitors that impair the binding of the E3 ligase to the oncosuppressor protein p53 favoring cell death ,137,138.C-Terminus (HECTs) E3 ligases is a smaller family of E3 ligases that are involved in a wide range of pathologies such as neurodegenerative disorders, hypertension, and cancer. This type of E3 ligases carry out a two-step ubiquitination mechanism: they first bind the Ub-E2 ligase and catalyze the transfer of the Ub onto a catalytic cysteine; secondly, they bind the substrate protein and transfer the Ub molecule. This type of mechanism voids the substrate specificity of the E2 ligases as the ubiquitination of the substrate protein will be directed by its interaction with the HECT ligase through adaptor proteins. Interestingly, HECT ligases can be hijacked by viral proteins that favor the ubiquitination of proteins that are not the natural substrates of this system [C-terminal end of the protein. This domain is comprised of two lobes connected by a flexible loop: the E2-binding domain at the N-lobe, and the C-lobe that contains the catalytic cysteine that binds to the Ub molecule [The Homologous to E6AP s system . The maimolecule . N-terminal domain: the NEDD4 subfamily that presents WW and C2 domains, the HERC subfamily that presents regulator of chromatin condensation-like domains (RLD); and the “Other” subfamily that have different N-terminal domains that may or may not contain the WW and C2 or the RLD domains [The HECT family can be divided into three subfamilies depending on the characteristics of the  domains . Interes domains . Inhibitors of this family of ligases have been identified using phage libraries, and despite not having a clear understanding of their mechanisms of action, they all alter the Ub transfer from the E2 ligase to the catalytic cysteine of the HECT domain . Smurf1 and Smurf2 are the two members of the Smurf (SMad Ubiquitination-Related Factor) family. The cellular location of the two Smurf family members will mainly depend on their target proteins. Interestingly, Smurf1 is mainly located in the cytoplasm, but can translocate to the nucleus, while Smurf2 acts in the opposite way; it is mainly nuclear but can translocate to the cytoplasm . In normHowever, Smurf2 can either act as tumor promoter or suppressor, depending on various factors such as tumor stage and type, amongst others . Smurf2 The tumor suppressor activity of Smurf2 is related to the ability of Smurf2 to induce cell senescence when telomers are shortened, thus avoiding the accumulation of mutations by degrading Id1 (Inhibitor of DNA binding 1), which leads to the expression of p16 that is the ultimate senescence inducer. Additionally, Smurf2 degrades: RNF20 (RiNg finger protein 20) that is responsible for histoneH2B ubiquitination; TopoIIα (Topiisomerase IIα) that is involved in chromosomal translocations; SATB1 (AT-rich Sequence-Binding protein 1) that promotes the growth and metastasis of colon cancer; KLF5 (Transcription factor-Krüppel-like factor 5) that promotes colon and bladder cell proliferation; and YY1 (Yin Yang 1) that is a Krüppel-like zinc finger transcriptional factor expressed in various cancers . N-terminal end, several WW domains, and the HECT domain at the C-terminal end [From a structural point of view, the Smurf subfamily belongs to the NEDD4 subfamily of HECT E3 ligases, as both Smurf1 and Smurf2 present the C2 domain at the inal end . N-terminal end, three WW domains , and the HECT domain at the C-terminal end. The C2 domain presents two main activities: on the one hand, it is responsible for the auto-inactivation of Smurf2 by interacting with the HECT domain [Structurally, Smurf2 presents the C2 domain at the T domain ,151; on T domain . The WW T domain . The intT domain ,150. N-Myc is Lys48-poliubiquitinated by HUWE1 in neuroblastome cells and in embryonic stem cells, thus allowing neuronal differentiation and cell cycle arrest [c-Myc, as the absence or mutation of this E3 ligase results in an observable increase in cell proliferation as a result of increased c-Myc levels [c-Myc, which led to the activation of its transcriptional activity and, in parallel, it also formed Lys48-poliubiquitin chains in MZ1 to induce its degradation. MZ1 is a component of the MYC/Max/MZ1 repressor complex [c-Myc in multiple myeloma, where it can be found overexpressed [HUWE1 is a two-faced ligase with respect to cancer, as depending on the type of cancer it will act either as an enhancer or a tumor suppressor ,152. Wore arrest ,156. Myac levels . On the  complex . In addixpressed . Anotherxpressed . MoreoveHUWEI has also been found to be implicated in an intricated p53-regulating pathway that involved different types of E3 ligases, tumoral suppressors and HDAC2. In fact, p53 and HDAC2 are two targets of this ligase ,153. p14N-terminal end is still being studied. In 2018, Jasper Sliumer and Ben Distel described the N-terminal end to have four domains: ARM (ARMadillo-type fold domain) which is believed to mediate in vesicular transport of many proteins; the UBA domain (UBiquitin Associated domain) which is responsible for binding polyubiquitin chains; the WWE domain (Triptophan and Glutamic acid-rich domain) which is responsible for protein–protein interactions during the ubiquitination phase; and the BH3 domain (Bcl-2 Homology region 3) which specifically recognizes the Bcl2-family member Mcl-1 [C-terminal end of the BH3 domain, they indicated the presence of the UBM, the function of which is yet to be fully elucidated [N-terminal end. According to this study, this end is made up of four Armadillo Repeated Liked Domains (ARLD1–4); a WWE domain; and a “Tower” and HWA (HUWE1 WWE module Associated) domain [HUWE1 belongs to the “Other” subfamily of HECT E3 ligases whose er Mcl-1 ,160,161.ucidated . Neverth) domain . The ARL) domain . Moreove) domain . N-terminal end interacts with the HECT domain, HUWE1 can either do so in a similar way or by oligomerization. The structural basis of the inhibition of HUWE1 is quite convoluted. The C-terminal domain has been found crystalized, forming an asymmetric dimer, but a small amino acid sequence before the C-terminal domain is responsible for the inhibition impairment of HUWE1. Additionally, tumor suppressor p14ARF works as a HUWE1 inhibitor by interacting with that small amino acid segment and hijacking the enzyme in the auto-inhibited dimer conformation [Interestingly, when other HECT family members present an inhibited conformation in which one of the domains in the ormation ,154,163.CRBN, cIAP1, MDM2, and CRL2VHL, as they present known ligands that can be functionalized to add the needed linker and POI ligand  a linker and a cap group (CG). The ZBG targets the catalytic zinc ion, while the linker establishes interactions with the amino acids lining the sides of the access tunnel to that ion; and, the cap group interacts with the surface of the protein . Despite® (potent and selective HDAC6 inhibitor [® (pan-HDACi) [®-based PROTAC  of 7.1 nM in MM.1S cells [However, despite the amount of described HDACis, only a few have been used as POI ligands in the design of HDAC targeted PROTACs: Nexturastat Anhibitor ), Crebinn-HDACi) , CI-994,n-HDACi) . The desn-HDACi) ,75,168, n-HDACi) ,168. Addn-HDACi) . A similbstrates . As a re50 of 42 nM [Selective, long-lasting, but reversible HDAC3 dose-dependent degradation was achieved by PROTAC V  in MDA-Mof 42 nM ,75. Inteof 42 nM . PROTAC It is noteworthy to highlight that, in the attempt to design a cIAP1-based HDAC directed PROTAC using SAHA as HDAC binding moiety, and bestatin as E3-ligase recruiter, instead of achieving PROTAC degradation, the resulting compounds proved to be strong dual inhibitors. Bestatin is also an approved AminoPeptidase N (APN) inhibitor, and the resulting hybrid molecules showed a greater inhibitory activity against APN than bestatin. Moreover, the inhibitory activity against HDACs 1, 6, and 8 of the hybrids was better than that of SAHA. Decreased HDAC protein levels were found in cellular assays, but this depletion was not due to the PROTAC effect of the hybrid molecules, and was similar to that found when using SAHA .VHL, the length of the PROTAC linker should be longer than if the ligase is CRL4CRBN [Selective HDAC inhibition has become a popular topic in drug discovery, as HDACs are implicated in the development of many types of cancer, diabetes, and inflammatory, neurodegenerative, and cardiovascular diseases. In recent years, many researchers have attempted to address this challenge by the design and synthesis of HDAC-directed PROTACs. The PROTAC strategy requires less selectivity and/or potency to produce HDAC degradation than classical inhibitors. PROTAC I  can induCRL4CRBN . In addiCRL4CRBN . In factCRL4CRBN .This review has attempted to shed light onto the structure of the different E3 ligases that naturally control the HDAC protein levels in the cell, in order to pave the way for the design and synthesis of new HDAC-directed PROTACs.https://pubmed.ncbi.nlm.nih.gov, last accessed on 8 September 2021, Web of Science , or Scopus . The structural information of the different E3 ligases was obtained from the Protein Data Bank  and UniProt .The bibliographic search was carried out using the Google and Google Scholar search engines combined with different databases such as PubMed . The 3D images of the proteins were created using PyMOL , and all of the chemical structures were drawn using ChemDraw 19 (www.perkinelmer.com).The schematic representations were created using BioRender .


Clostridium perfringens enterotoxin (CPE)-mediated oncoleaking. This is achieved by CPE suicide gene therapy to treat PC, which overexpresses the claudin-3 and -4 (Cldn3/4) tight junction proteins, which are targets of CPE action. This targeted gene therapy causes rapid eradication of Cldn3/4 overexpressing PC cells via oncoleaking and initiation of apoptotic/necrotic signaling. We demonstrate efficacy of this approach in vitro and after nonviral in vivo gene transfer in cell lines and in patient derived xenograft PC models. This therapy approach has translational potential for treatment of pancreas carcinomas and could also be translated into new combination settings with conventional chemotherapy.Current therapies for pancreas carcinoma (PC) are of limited efficacy due to tumor aggressiveness and therapy resistance. Bacterial toxins with pore-forming (oncoleaking) potential are promising tools in cancer therapy. We have developed a novel, suicide gene therapy treatment, based on Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options.  Pancreatic cancer (PC) is one of the leading causes of cancer death in developed countries and one of the most lethal malignancies worldwide ,2. PC haClostridium perfringens enterotoxin (CPE) as well as their encoding gene sequences translated into toxin-based cancer therapy [Apart from all the established treatment modalities, gene therapy might be an alternative for an effective PC therapy. In particular, suicide gene therapy with use of bacterial toxins is attractive. Bacterial toxins are produced and released by bacteria affecting different targets in host cells. An increasing number of in vitro and in vivo studies using different bacterial toxins demonstrate efficacy in cancer cell killing . During  therapy ,12,13,14Clostridium perfringens [2+ influx and consequently to necrotic cell death. Low CPE concentration, however, results in low pore numbers, rather leading to apoptotic cell death [CPE is of particular interest, since it allows effective and targeted attack of cancer cells. The toxin is produced by the Gram-positive, anaerobic bacterium fringens . This prfringens ,17 and tfringens ,19,20,21ll death .Numerous reports showed that compared to normal tissues, particularly epithelial cancers, such as pancreatic, colon, ovarian, breast and prostate cancer possess high Cldn3/4 expression and dysregulation ,24,25. BFor comprehensive and detailed description of all methods see 2. The identity of all cell lines was confirmed by STR-genotyping .Human pancreas carcinoma AsP-1, BxPC-3, Capan-1 and Sk-Mel-5 melanoma cells were grown in RPMI medium , 10% FCS . The human pancreas carcinoma HUP-T3, MIA PaCa-2, PA-TU-8902 and human colon carcinoma HT-29 cells were grown in DMEM (Gibco), 10% FCS (Biochrom). All lines were kept at 37 °C, 5% COFor in vivo studies, 21 pancreas carcinoma PDX models of stage IV , were used .Total RNA was isolated using GeneMatrix Universal RNA Purification Kit EURx  and reverse transcribed. Quantitative real-time PCR (qPCR) was performed with SYBR GREEN in the LightCycler 480 . They following primers were used for claudin-3: forward 5′-CTGCTCTGCTGCTCGTGTCC-3′; reverse 5′-TTAGACGTAGTCCTTGCGGTCGTAG-3′; for claudin-4: forward 5′-CCTCTGCCAGACCCATATAA-3′; reverse 5′-CACCGTGAGTCAGGAGATAA-3′; for RNA polymerase II: forward 5′-GCACCACGTCCAATGACA T-3′, reverse 5′-GTGCGGCTGCTTCCATAA-3′. Normalization was done with the housekeeping gene glucose-6-phosphate dehydrogenase (G6PDH) using the hG6PDH Roche Kit (Roche Diagnostics). Primers used for G6PDH: forward 5′-GAAGATGGTGATGGGATTTC-3′, reverse 5′-GAAGGTGAAGGTCGGAGT-3′.2O). For isolation of membrane, cytosolic and nuclear fractions the Cell Fractionation kit  was used. Lysates were electrophoresed in 10% precast NuPAGE gels  and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in TBS  and washed in TBST (0.05% Tween 20 in PBS). Western blot was performed with primary rabbit anti-claudin-3 antibody , rabbit anti-claudin-4 antibody (Acris), rabbit polyclonal anti-CPE , mouse monoclonal anti-β-tubulin  or mouse monoclonal anti-β-actin antibody . As secondary HRP-labeled goat anti-rabbit-IgG antibody , HRP-labeled goat anti-mouse-IgG antibody  or goat anti-mouse IgM/IgG  was added. Detection was done using ECL solution  and exposure to Kodak X-Omat AR film .Cells or tissue cryosections were lysed in RIPA buffer  and the pcDNA3-CPE-GFP (CPE-GFP) plasmids were used ,30. Prep5–5 × 105 cells/well were seeded in 6-well plates and after 24 h transfected with respective plasmid DNA plus transfection reagent  according to manufacturer’s instruction. After 12–72 h, cells were washed with PBS and RNA or protein was isolated.For transfection experiments, the pCpG-optCPE (optCPE) or the pCpG-mcsG2 empty vector control (VC) plasmid  was used. A mutated optCPE construct unable to bind Cldn3/4 . Cells were washed with PBS 48 h or 72 h post transfection and RNA and protein was isolated.Knockdown of Cldn3/4 was done with pools of three different iBONi short interfering RNAs (siRNA) for Cldn3 or 4. For siRNA knockdown, 3 × 103–4 × 105 cells were seeded into 96-well plates and 24 h later 0, 50, 100, 150 ng mL−1 recCPE was added and incubated for 72 h. For determination of toxicity of released CPE in supernatants of transfected cells, 6 × 103 non-transfected cells were seeded into 96-well plates. After 24 h, 100 µL of supernatants from optCPE-transfected cells were added to respective non-transfected cells and incubated for 72 h. For all cytotoxicity assays, 5 mg mL−1 MTT -2,5-diphenyltetrazolium bromide; Sigma) was added 72 h after CPE incubation. Absorbance was measured in triplicates at 560 nm in a microplate reader  and values are expressed as percentage of untreated controls.MTT assay was used to test cytotoxicity of recombinant CPE (recCPE) or of optCPE transfection and to test cytotoxicity of released CPE from transfected cells. For sensitivity testing of cell lines towards recCPE 6 × 10Clostridium perfringens enterotoxin ELISA  was performed. For this, 4 × 105 cells were seeded into 6-well plates and transfected with pCpG-optCPE or pCpG-mscG2 plasmid-DNA. Recombinant CPE was used as standard at serial dilutions of 0.4–25 ng CPE mL−1. Measurements were done in duplicates at 450 nm in the microplate reader (Tecan) and values are expressed as percentage of untreated controls.For quantification of CPE in supernatants 24 h or 48 h after transfection, Ridascreen 5 cells were seeded into 4-well chamber slides and after 24 h washed with PBS, fixed in 4% paraformaldehyde  in PBS for 15 min, permeabilized with 0.5% Triton-X in PBS for 10 min and blocked with 1% IgG-free albumin  and 0.05% Tween-20 in PBS for 1 h. Rabbit anti-human Cldn3 or anti-human Cldn4 antibody (Acris) was added as primary antibody, cells were washed with TBST, then incubated with HRP-conjugated goat anti-rabbit IgG antibody , washed again and incubated with diamino-benzidine . Cells were then washed in ddH2O, counterstained with hemalum (Roth) and evaluated in a light microscope .For immunohistochemistry, 2 × 105 cells were seeded onto cover slips , washed, fixed in 4% PFA, quenched with 0.1 M glycine for 20 min and blocked with 1% serum-free albumin, 0.05% Tween-20 for 1 h. The respective primary goat anti-claudin-3 rabbit polyclonal IgG , goat anti-claudin-4 rabbit polyclonal IgG  anti-CPE rabbit polyclonal IgG, (BioRad) was added, cells were washed with TBST and incubated with secondary antibody . Nuclei were stained with DAPI (Sigma-Aldrich), counterstained with Alexa 555-phalloidin (Thermo Fisher Scientific) and evaluated in a confocal fluorescence microscope (Zeiss).For immunofluorescence, 2 × 102O2 for 10 min, washed with PBS, permeabilized by 0.2% Triton X-100 and blocked with 1% IgG-free albumin and 0.05% Tween-20 for 1 h. Steps for staining are similar to aforementioned procedure.For analysis of Cldn3/4 expression or CPE expression after gene transfer in the PDX tumors, 3–5 μm thick paraffin embedded (FFPE) tumor sections were deparaffinized, fixed with 4% PFA, quenched with 0.1 M glycine for 20 min, incubated with 3% H6 cells or pieces of approximately 3 × 3 mm in size of patient derived pancreas cancer PDX tissue were inoculated into the left flank of female NMRI: nu/nu mice . Animals were randomized at tumor volume of 0.3 cm3 and intratumoral non-viral in vivo jet injection gene transfer  was performed in anesthetized animals [−1 PBS in 10 µL injection volume). Tumor volumes (TV) were measured at indicated time points and calculated using the formula: TV = (width2 × length)/2. Animals were sacrificed and tumors were harvested for further analysis. Body weights, clinical signs and behavior were recorded for all mice twice a week. All experiments were performed in accordance with the UKCCCR guidelines and approved by local authorities .For establishment of subcutaneous tumors, 1 × 10 animals . For thit-test and one way-ANOVA test were used. For the statistical analyses of in vivo experiments the non-parametric, unpaired t-test was used. Error values for the in vitro experiments are S.D. and for in vivo experiments S.E.M.For statistical analyses of the in vitro experiments the Student’s Prerequisite for targeted antitumoral oncoleaking activity of CPE is presence of high affinity CPE receptors, such as Cldn3/4, as the CPE-mediated cytotoxicity requires their accessibility. This was analyzed in a panel of human PC cell lines at mRNA and protein level. By this, Capan-1, PA-TU-8902, AsPc-1 and the positive control human colon cancer HT-29 control cells showed high Cldn3/4 mRNA levels, whereas moderate levels of Cldn3/4 were detected in MIA PaCa-2 and HUP-T3 cells A. BxPC-3−1 were applied for 72 h and cytotoxicity was measured (p = 0.0001) dose dependent sensitivity toward recCPE. Despite high Cldn3/4 expression level, PA-TU-8902 cells showed comparatively low sensitivity toward recCPE, since the highest concentration reduced cell viability only to 60% (p = 0.0019). By contrast, in BxPC-3 and MIA PaCa-2 cells with low Cldn3/4 expression, higher cytotoxicity was measured. In these cells viability was significantly (p = 0.0001) reduced to 40% or 32% compared to control, respectively. However, Cldn3/4 negative cell line SK-MEL-5 was insensitive towards the toxin. RNAi-mediated downregulation of Cldn3/4 expression in MIA PaCa-2 and HUP-T3 cells transfected with siCldn3 and siCldn4 or siCtrl, abolished sensitivity toward recCPE (For sensitivity testing of Cldn3/4 expressing pancreatic cancer cell lines toward recombinant CPE (recCPE) protein, increasing recCPE concentrations from 0 to 250 ng mLmeasured D. High Cd recCPE . All thiTo evaluate antitumoral efficacy of CPE gene transfer, first presence and expression kinetic for CPE was analyzed. After gene transfer, the immune fluorescence in Capan-1, HUP-T3 and PA-TU-8902 cells showed strong cytoplasmic staining for CPE A. Furthep < 0.001; To further prove selectivity of CPE cell killing, RNAi experiments were performed for specific downregulation of Cldn3/4 expression. MIA PaCa-2 and HUP-T3 cells were transfected with a pool of siCldn3 and siCldn4 or siCtrl, respectively D. The do2+ influx due to pore formation, activation of caspase-3 and binding of Annexin-V to apoptotic cells. Furthermore, lactate dehydrogenase (LDH) release and calpain activation was determined.For the improved and more effective use of the pore-forming toxin, the mode of cell death by CPE gene therapy was of interest. We determined timely activation of apoptotic or necrotic pathways in optCPE-transfected pancreatic cancer cells by live cell imaging for apoptotic signaling, CaThe first indication for optCPE action is uncontrolled calcium influx. In MIA PaCa-2 and PA-TU-8902 cells optCPE gene transfer induced rapid calcium influx indicating rapid pore formation between 8 and 14 h after gene transfer, although to different extents in the two lines  induced strong cytotoxicity. The lower toxin amount, released by MIA PaCa-2 cells  resulted in lower cytotoxicity.The transfection experiments revealed much higher optCPE toxicity than anticipated by transfection rates of 10–65% of the human PC cell lines. This suggests that non-transfected cells are potentially affected by a bystander effect of released optCPE. To prove this, supernatants of optCPE-transfected cells were examined. First, we analyzed cytotoxicity of the supernatants released 24 h, 48 h and 72 h after optCPE transfection of PA-TU-8902 and MIA PaCa-2 pancreas cancer cells A. By thi−1 in supernatant of transfected MIA PaCa-2 and PA-TU-8902 cells and 242.6 and 344.5 ng mL−1 CPE in exosomes, respectively  and tetraspanin CD63 in the Western blot B. Both, ectively C. This s−1 at 24 h to 290 ng mL−1 optCPE after 96 h (−1 optCPE), which decreased over time to 2–14 ng mL−1 optCPE  and tumor volume (p < 0.0001) compared to vector control (VC) treated mice  or VC (p = 0.0003)-transfected tumors (p < 0.00001) and mutCPE (p = 0.0004)-transfected tumors. The mutCPE-mediated effects were not different from the VC-transfected group. This validates the strict Cldn3/4 selectivity of optCPE cytotoxicity after intratumoral in vivo gene transfer as important feature for targeted gene therapy.To address selectivity of in vivo optCPE action, an expression plasmid with mutated optCPE  or VC (p = 0.0009)-transfected tumors. This was particularly seen in areas, with close vicinity to necrotic areas, which could partially be explained by the optCPE-mediated bystander effect  compared to VC-transfected tumors (p = 0.014) compared to VC treated tumors (p = 0.0015), particularly for areas in close vicinity to necrosis (p = 0.0002) compared to VC treated control tumors  was significantly reduced in these tumors compared to VC  is one of the most lethal solid malignancies worldwide . DespiteClostridium perfringens enterotoxin; CPE) revealed their capability for effective cell killing [An increasing number of in vitro and in vivo studies, which use bacterial toxins for cancer treatment (e.g., diphtheria toxin, streptolysin O or  killing ,14. The  killing ,39. The  killing . Elevate killing ,44,45,46 killing . This is killing ,47,48,49Structure-based mutagenesis enabled design of CPE mutants that also target claudins such as Cldn1 or -5 that are not high affinity receptors for CPE wildtype. Local injection of these recombinant CPE mutants strongly reduced growth of Cldn1-expressing thyroid tumors in CDX models . CPE mut2+ influx, activation of caspases and calpain. This suggests, that CPE gene transfer induces different cell death mechanisms, known as “apoptosis-necrosis continuum” [2+ influx [Here we focused on the mechanisms mediated by CPE gene transfer for suicide gene therapy. Our analyses revealed that CPE gene transfer induced rapid fragmentation of nuclei, massive cell swelling, membrane blebbing and formation of membrane vesicles, release of LDH, increased Cantinuum” , which a+ influx ,51, whic+ influx ,52,53. D+ influx ,30. Our For efficient cell killing the bystander effect is supportive in gene therapy. Our immunofluorescence analysis in optCPE-transfected cells revealed vesicle like accumulations as indication for exocytosis. Further, membrane budding was observed, representing a key step in vesicular transport, multivesicular body and exosome formation. Indeed, we showed that CPE is released from cells and is also liberated via exosomes after CPE gene transfer and is acting on neighboring cancer cells. The recent study of Shrestha and colleagues also confirmed a bystander killing mechanism of native and recombinant CPE protein . They deOur study evaluated the anticancer potential of CPE gene therapy in different luciferase-expressing CDX and PDX models. Such locally applied toxin-based gene therapy has been successfully tested for diphtheria toxin in human PSA positive prostate xenograft tumors . In our In this study, we also evaluated the potential of CPE gene transfer in combination with drug treatments. In this context, we determined a drug treatment-induced increase of Cldn3/4 expression. Due to this unexpected result, we performed combination studies with gemcitabine and CPE gene transfer, which demonstrated a synergistic effect of this combination. This implied that gemcitabine can induce Cldn expression, which in turn leads to an improved target availability for CPE gene therapy as a basis for new combination approaches in PC therapy.In summary we established the efficient use of oncoleaking optCPE suicidal gene therapy in vitro, and more importantly, in subcutaneous and orthotopic human pancreatic cancer CDX and PDX models in vivo. The study emphasizes the great potential of this novel strategy, as it is selective for therapy of pancreas carcinoma.