Rutin has been reported as a potential anti-cancer agent for several decades. This study evaluated the effects of rutin on the proliferation, metastasis, and angiogenesis of MDA-MB-231 and MCF-7 breast cancer cell lines. Increasing concentrations of rutin significantly stimulated the proliferation of MDA-MB-231 and MCF-7 cells compared to controls. Wound scratch assay demonstrated that rutin had an inducing effect on the migration of the cells. In MDA-MB-231 and MCF-7 cells, rutin upregulated MKI67, VIM, CDH2, FN1, and VEGFA and downregulated CDH1 and THBS1 genes. It also increased N-cadherin and VEGFA and decreased E-cadherin and thrombospondin 1 protein expression. Our data indicated that rutin could stimulate proliferation, migration, and pro-angiogenic activity in two different breast cancer cell lines. This phytoestrogen induced invasion and migration of both cell lines by a mechanism involving the EMT process. This suggests that rutin may act as a breast-cancer-promoting phytoestrogen. Breast cancer is the most common cause of female mortality from cancer around the world . Based oClassical methods of breast cancer therapy such as surgery, radiotherapy, hormone therapy, and chemotherapy are available in clinical settings ,5. HowevMetastasis is a multi-step process during tumor development leading to the spread of cancer cells to the secondary organs or tissues . Breast Rutin is a flavonoid compound found in a variety of plant sources. This compound has various medicinal effects, including anti-inflammatory, antioxidant, anti-diabetic, vascular protective, antimicrobial, and anti-cancer activities . The antIn breast cancer cell lines, data on rutin are more confusing. Indeed, one study performed on MB-MDA-231 and MCF-7 cells has shown that rutin does not possess anti-proliferative effect on these cells, at least up to 20 µM, but was able to increase the cytotoxicity of two chemotherapy agents, cyclophosphamide and methotrexate . On anot2. Rutin was purchased from Sigma-Aldrich  and the stock solution was prepared in dimethyl sulfoxide (DMSO).The breast cancer cell lines used in this present study, MCF-7 and MDA-MB-231, were purchased from Pasture Institute, Tehran, Iran. All the materials used for cell culture were purchased from Gibco, UK. MCF-7 and MDA-MB-231 cell lines were cultured and incubated in a humidified incubator at 37 °C and 5% CO4 cells per well and allowed to adhere to the bottom of the wells. After 24 h, the seeded cells were treated with increasing concentrations of rutin  for 24, 48, and 72 h. The control cell lines were treated with normal culture medium and vehicle (DMSO). Then, the viability of each cell line was assayed using the 3--2,5-diphenyltetrazolium bromide (MTT) method. Briefly, at the end of the treatment period, the cell lines were incubated with 5 mg/mL MTT solution for 4 h. Subsequently, the MTT solution was discarded and the formazan product formed was solubilized with 100 µL of DMSO. After 15 min, the absorbance was read at 570 and 630 nm using a plate reader spectrophotometer .Cells were seeded into 96-well cell culture plates at the density of 105 cells were seeded in a 6-well cell culture plate and allowed to adhere and grow to 50–60%. Subsequently, two horizontal scratches were created in the center of each well using 200 µL sterile pipette tips. One vertical line was created in each well to take photographs of scratches at the same point. Then, each well was washed with phosphate-buffered saline (PBS) buffer solution (pH~7.4) to remove cell debris. Afterward, three test wells were treated with 200 and 400 μM rutin compound and three control wells were treated with a normal culture medium. Finally, the photographs of the wound-healing capacity of both cell lines were captured at 0 h, 9 h, and 24 h.The effect of rutin treatment on the invasiveness and migratory capacity of MCF-7 and MDA-MB-231 cell lines was evaluated by in vitro scratch assay as previously described . Briefly−ΔΔCT formula. The effect of rutin on the gene markers of proliferation, EMT, and angiogenesis was evaluated by using the Quantitative Real-time PCR technique. Following the treatment of MDA-MB-231 and MCF-7 cell lines with rutin at the concentration of 200 µM for 48 h, the total RNAs were extracted from treated and control cells by an RNeasy mini kit . To assess the quality of total RNA, the absorbance of the samples was measured by a Nanodrop 2000c spectrophotometer. Afterward, 2 μg of total RNA sample was collected to synthesize cDNA molecules using a cDNA Synthesis Kit . Then, ABI PRISM7900HT  qRT-PCR detection system with SYBR GREEN PCR master mix  was used to assess the expression levels of MKI67, CDH1, CDH2, VIM, FN1, VEGFA, and THBS1 genes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was considered the internal control gene. Relative expression (fold changes) of the above-mentioned genes was calculated using the 25 cells/well and allowed to adhere overnight. Then, the test cell lines were treated with 200 µM rutin for 48 h. The control cell lines were treated with culture medium. After 48 h, the treated cells were lysed using radioimmunoprecipitation assay (RIPA) buffer . The protein content of the whole-cell lysates was measured using the Bradford method [Cells were seeded in 6-well culture plates at a density of 2 × 10d method . Using Sp-values less than 0.05 and 0.01 were considered significant. All data are representative of the mean ± standard deviation (SD). Analysis of wound areas was performed by ImageJ software .The results of the present study were obtained from three independent experiments. The statistical analyses were performed with GraphPad Prism software version 7. The data were analyzed using the one-way analysis of variance (ANOVA) test followed by Duncan’s multiple range tests as the post hoc. Treatment of MDA-MB-231 and MCF-7 cell lines with increasing concentrations of rutin for 24, 48, and 72 h promoted the proliferation of both cancer cell lines in a dose-dependent manner in comparison to the culture medium (control) and DMSO-treated cells. As depicted in To determine the effect of rutin on the invasion and migration of MDA-MB-231 and MCF-7 cell lines, the cells were seeded into 6-well plates and treated with 200 and 400 μM rutin. The results showed that rutin treatment promoted the invasion and migration of MDA-MB-231 cells at both concentrations of this natural compound A. IndeedSimilarly, in MCF-7 cells, rutin had a promoting effect on the invasion and migration of the treated cells at concentrations of 200 and 400 μM in comparison to untreated controls A,B. HoweTo evaluate the effect of rutin on markers of proliferation, EMT, and angiogenesis, the expression levels of the corresponding gene markers in the MDA-MB-231 and MCF-7 cell lines were quantified. Treatment of the MDA-MB-231 and MCF-7 cell lines with rutin had a modulatory effect on the expression levels of various proteins involved in the angiogenesis and EMT process in these cancer cells. Thus, according to the results of Western blotting analyses, rutin, at the concentration of 200 μM, had a possible effect on the EMT process in the MDA-MB-231 cell line by downregulating E-cadherin and upregulating N-cadherin in comparison to untreated control cells. However, it had no remarkable effect on the expression levels of Vimentin and Fibronectin 1 proteins compared with the controls. This natural compound also showed no significant effect on the protein levels of proliferation marker ki67 in the MDA-MB-231 cell line. Nonetheless, rutin activated angiogenesis by elevating the expression of the pro-angiogenic marker VEGFA and declining the anti-angiogenic marker Thrombospondin 1 B.Treatment of MCF-7 cells with rutin 200 µM decreased the protein expression of E-cadherin with increased levels of ki67, N-cadherin, and Fibronectin 1 protein compared with the untreated control with no significant effect on the protein level of Vimentin B. As obsAccording to the literature, rutin has shown anti-proliferative and anti-metastatic effects in some cancer cell lines . For breIn the present study, the MTT assay results revealed that rutin stimulated the proliferation of both MDA-MB-231 and MCF-7 cancer cell lines at concentrations greater than 200 µM. Evaluation of the expression of ki67 also uncovered that rutin increased this marker in the MDA-MB-231 cell line at the mRNA level but had no effect on the expression of its protein product. Rutin upregulated mRNA and protein levels of ki67 in the MCF-7 cell line. Ki67 is known as a key marker of tumor proliferation and invasiveness [The results of the wound scratch assay illustrated that rutin acts as an inducer of invasion and migration in the MDA-MB-231 cell line at doses of 200 and 400 µM. However, significant wound closure was observed in MCF-7 cells only after treating the cells with rutin 400 µM. This difference may be due to the higher migratory activity of the MDA-MB-231 cell line in comparison to MCF-7 cells. The wound-closure capacity of rutin may propose a potential metastasis-inducing activity of rutin in breast cancer cells.Based on the results of the wound scratch assay, we decided to uncover the mechanism by which rutin has promoted the metastasis of MDA-MB-231 and MCF-7 breast cancer cells. Thus, the effect of rutin treatment on the expression levels of EMT markers in both cancer cell lines was evaluated. EMT is the leading event in the process of metastasis that allows an epithelial cell to undergo some biochemical changes leading to a mesenchymal cell phenotype with higher metastatic capacity and invasiveness . EMT is The present data are opposite to those reported in the literature. One possible explanation for these controversial results is based on the fact that rutin is a phytoestrogen compound with a variety of beneficial or adverse effects depending on the subject studied, the sex, the age, and the physiological status . PhytoesAn alternative scenario may arise from the intracellular metabolism of rutin in breast cancer cell lines. Harris et al. demonstrated that a variety of phytoestrogens act as potent inhibitors of sulfotransferases, which are enzymes that add a sulfate group to estrogens to inactivate them . MoreoveThe present data unraveled the cancer-promoting effects of rutin on two different breast cancer cell lines. Contrary to the previously published data showing the anti-proliferative effect of rutin on HGF-treated breast cancer cells, the present study demonstrates that this natural phytoestrogen stimulated the proliferation, invasiveness, and pro-angiogenic capacity of breast cancer cells. Rutin induced invasion and migration of these cancer cell lines by a mechanism that involved the EMT process. It also stimulated the angiogenic pathway involved in the metastasis of breast cancer cells. Although rutin has been reported as an anti-cancer agent in the literature, these data suggest that it could act as a breast-cancer-promoting or progressing natural compound. Although the present data is primary, it may pave the way to design more comprehensive studies for the evaluation of the effects of rutin phytoestrogen in a variety of cancers.


GBA1, which encodes the lysosomal hydrolase glucocerebrosidase (GCase), are a common risk factor for the neurodegenerative movement disorder Parkinson's disease (PD). Consequently, therapeutic options targeting the GCase enzyme are in development. An important aspect of this development is determining the effect of potential modifying compounds on GCase activity, which can be complicated by the different methods and substrate probes that are commonly employed for this purpose.Heterozygous mutations in in situ in the lysosome.In this study, we employed the GCase substrate probe 5-(pentafluorobenzoylamino)fluorescein di-D-glucopyranoside (PFB-FDGlu) in combination with live cell imaging to measure GCase activity GBA1 knockout neural cells and was then used to assess GCase activity in iPSC differentiated into neural stem cells and neurons that were obtained from idiopathic PD patients and PD patients with the LRRK2 G2019S and GBA N370S mutations, as well as controls (n = 4 per group). Heterogeneity in GCase activity was observed across all groups. However, a significant inverse correlation between GCase activity and levels of alpha-synuclein protein was observed.The live cell assay was validated using the GCase inhibitor conduritol-B-epoxide and with The live cell imaging assay for GCase activity could be useful for further understanding the role of GCase in PD and screening potential modifying compounds in differentiated human cell models. GBA1, may be associated with up to 15% of cases of the neurodegenerative movement disorder Parkinson's disease (PD) , encoded by After synthesis in the endoplasmic reticulum, GCase is trafficked to the lysosome in association with lysosomal integral membrane 2 (LIMP2) fluorescein di-D-glucopyranoside (PFB-FDGlu). The PFB-FDGlu probe enters the cell via pinocytosis and travels to the lysosome, where it is metabolized by GCase to release fluorescein , the PFB-FDGlu GCase assay was modified in this study to work with live cell imaging instead of flow cytometry. We demonstrated the specificity of the assay using the GCase inhibitor conduritol b-epoxide and GBA1 knockout murine neural cells and assessed GCase activity in neural stem cells and neurons differentiated from iPSC obtained from patients with PD-associated mutations LRRK2 G2019S and GBA1 N370S, as well as iPSC from PD patients without these mutations and from controls. Heterogeneity in GCase activity was evident across the cell lines, but a significant inverse correlation between GCase activity and alpha-synuclein levels was measured in the differentiated neurons. The live cell imaging protocol using PFB-FDGlu could, therefore, be useful for understanding how GCase activity contributes to PD pathogenesis and/or screening for modulators of GCase activity in neural cells in vitro, and results from the differentiated neurons add to the evidence that GCase contributes to the regulation of alpha-synuclein turnover.To assess 4 cells/well and maintained as described in a previous study  immortalized neural cells were kindly provided by Ellen Sidransky from the National Institutes of Health, and the generation and characterization of these cells have been described previously  pre-coated with poly-L-lysine and incubated at 37°C with 5% CO2 until confluent.Human neuroblastoma SH-SY5Y cells were maintained in DMEM/F-12 supplemented with 10% low-endotoxin fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin . For experiments, cells were plated on clear bottom 96-well plates  at 6 × 10GBA1 N370S mutation and matched controls were obtained from the Golub Capital iPSC Parkinson's Progression Marker Initiative (PPMI) sub-study (www.ppmi-info.org/cell-lines). The investigators within PPMI contributed to the design and implementation of PPMI and/or provided data and collected samples but did not participate in the analysis or writing of this report. For up-to-date information on the PPMI study, please visit www.ppmi-info.org. The heterozygous LRRK2 G2019S iPSC lines, tone control iPSC line, and one idiopathic PD iPSC line were generated in-house by reprogramming fibroblasts obtained from the Coriell Cell Repository, and the generation and characterization of all these lines have been described previously  from PD patients with and without a heterozygous 4 cells/well and maintained as described for 24 h to allow the cells to attach. For neuronal differentiation, NSCs were plated on clear bottom 96-well plates  coated with 10 mg/mL poly-L-ornithine  and 10 μg/mL laminin  at a density of 1 × 104 cells/well. NSCs were then differentiated into neurons over 21 days using neurobasal plus medium supplemented with 2% B-27 serum-free supplement, 2 mM L-glutamine, and 1% penicillin-streptomycin , as we have described previously  were differentiated from the iPSCs using the Gibco™ PSC Neural Induction Medium kit  as per the manufacturer's instructions. NSCs were plated on Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix  and cultured in neural expansion medium composed of 50% advanced DMEM and 50% neurobasal medium supplemented with neural induction supplement and 1X penicillin-streptomycin . The medium was changed every 2 days. NSCs were detached with Accutase  and passaged in the presence of 10 μL/ml RevitaCell supplement  when they reached 80% confluence. For experiments, NSCs were plated on clear bottom 96-well plates  coated with Gletrex  at 2 × 10490)/maximum LDH release (OD490))*100.Lactate dehydrogenase (LDH) present in the supernatant was measured using the CytoTox96 non-radioactive cytotoxicity assay  following manufacturer instructions before and 2 h after imaging for GCase activity. A 1% Triton-X100  solution was used as a positive control for maximum LDH release. Cell survival percentage was calculated as  and fixed with 4% paraformaldehyde at room temperature for 15 min. Cells were then washed twice with 1 X PBS and permeabilised with 0.3% Triton X100  in 1 X PBS for 15 min, followed by blocking with 3% bovine serum albumin in 1 X PBS at room temperature for 1 h. Incubation with primary antibodies  was performed in blocking buffer at 4°C overnight. We previously demonstrated the specificity of the alpha-synuclein antibody using alpha-synuclein knockout SH-SY5Y cells  , 400 nM of Bafilomycin , or an equivalent volume of DMSO for 1 or 4 h for bafilomycin. Following this, 20 nM Hoesch33342 (Bio-Rad PUREBLU™ #1351304) and 75 mM GCase substrate PFB-FDGlu [5(Pentafluorobenzoylamino) Fluorescein Di-β-D-Glucopyranoside]  were added. Plates were then rapidly inserted in the high-content imaging system Opera Phenix Plus  and imaged at the indicated times. Images were taken using a 20X water immersion objective, and at least 8 fields of view were imaged per well. Every experiment was performed in triplicate, with three wells per condition each time. Images were analyzed using Harmony software following standard protocols. Briefly, nuclei were detected and counted using the inbuilt “Detect nuclei” feature; the green fluorescence signal was thresholded using untreated wells  to select the threshold value; and the MFI was calculated per field of view and divided by the number of nuclei present in the image to obtain MFI/cell values. The GCase activity ratio was calculated as follows: (MFI/cell of PFB-FDGlu-treated wells divided by the MFI/cell of PFB-FDGlu+CBE-treated wells). For colocalization experiments, designated wells were stained with Lysotracker Deep Red  at 75 nM for 45 min to allow the reagent to enter the cells and stain the lysosomal compartment. Following this, the medium was replaced with fresh media containing 20 nM Hoesch33342 and 75 mM PFB-FDGlu. Plates were then imaged at 30 min at 40X water immersion objective.post-hoc testing and adjusted p-values for multiple comparisons was used to compare effects between groups or individual cell lines. Significance was accepted at a p-value of < 0.05. A Spearman's correlation was performed to determine the association between GCase activity and alpha-synuclein.Statistical analysis was performed using GraphPad Prism version 9.0 for macOS. A one-way ANOVA with Bonferroni n = 8), prompting the consideration of live imaging where the cells would not need to be detached and centrifuged. Initial time course experiments in differentiated SH-SY5Y cells . Post-hoc testing correcting for multiple comparisons showed that iPD-2 and iPD-3 lines were significantly lower and higher than all control lines, respectively. GBA1-1 and GBA1-3 lines were significantly lower and higher than all control lines, respectively. All LRRK2 lines were similar to control lines, except for Control-1, which was higher than all LRRK2 lines and all other control lines . The cell lines iPD-2 and iPD-3 were significantly higher than all control cell lines except Control-3. The GBA1-1 cell line was significantly lower than Control-1 and Control-3. The LRRK2-2 line was significantly higher than the Control-2 and Control-4 lines (p = 0.579)  .p = 0.029)  . This maGBA1 comprise one of the strongest genetic risk factors for PD, there is much interest in understanding how mutations affect GCase function and how this enzyme could be therapeutically targeted. Fluorescent GCase substrate probes such as PFB-FDGlu (Mazzulli et al., GBA1 mutations on GCase activity and/or screening for modulators of GCase activity, with particular utility for assessing GCase activity in differentiated neuronal cell lines.As mutations in GBA1 mutations, and is associated with alpha-synuclein pathology (Gegg et al., A number of studies support a link between GCase activity and levels of the pathological PD protein alpha-synuclein. GCase activity is lower in postmortem brain tissues from PD patients, including idiopathic patients without n = 3 GCase N370S iPSC differentiated to dopaminergic neurons, overall levels of GCase protein were the same as control. However, there was a shift to a higher-molecular-weight form of GCase as a result of increased glycosylation (Fernandes et al., n = 3 GBA N370S iPSC differentiated to dopaminergic neurons, a significant decrease in GCase protein was measured in the mutation cell lines compared to controls (Yang et al., n = 2 PD patients with the LRRK2 G2019S mutation. In this study, there was an ~40% reduction in live cell GCase activity measured with the PFB-FDGlu probe (Ysselstein et al., Although an inverse correlation between alpha-synuclein and GCase activity was observed across all cell lines, it is also noteworthy that significant group effects due to the LRRK2 G2019S or GCase N370S mutations were not observed. Studies on heterozygous GCase N370S mutation iPSC-derived neurons have been performed before with mixed outcomes. In one study of In summary, we optimized a live cell imaging-based assay for lysosomal GCase activity that can be used to further explore and understand PD phenotypes in differentiated cell models, as well as for screening potential modifying compounds of GCase activity.The original contributions presented in the study are included in the article/AL-G: experimental work, data analysis, figure preparation, and manuscript drafting. SZ and SK: experimental work, data analysis, and figure preparation. LH: experimental work and data analysis. WK and GH: manuscript drafting and editing and experimental design. ND: conceived the idea, experimental design, funding, data analysis, figure preparation, and manuscript drafting and editing. All authors contributed to the article and approved the submitted version.


Articles were selected on PubMed, Google Scholar, LILACS, BVS and SciELO, using keywords “thyroid cancer”, “flavonoids” and “TPC-1”, until June 2022. 185 studies were selected. After identification and exclusion of duplicates and exclusion criteria applied, 11 original articles were evaluated. Of these, the findings of flavonoids added to TPC-1 were: inhibition of cell growth and viability, promotion of cell cycle arrest and induction of apoptosis. Polyphenolic compounds have antineoplastic properties by different mechanisms as shown in vitro, but the concentrations needed are above usual dietary consumption and the findings are limited to experimental cellular studies. Despite that, these results should be useful to guide further analysis aiming to reveal the real safety and efficacy of polyphenols in this scenario.Thyroid cancer usually responds to surgical and ablative therapy, but when it's refractory the alternative lies in tyrosine kinase inhibitors that, in addition to harmful side effects, acts only in a palliative way. The concern for other therapeutic possibilities brought evidence on flavonoids, hypothesizing a possible strategy. This review aimed to organize a compilation of  RET/PTC1 (rearranged during transfection/papillary thyroid carcinoma type 1) rearrangement and BRAF V600E  mutations are present   . The objA systematic review of the literature was carried out, designed, structured and registered based on the items of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) tool, and registered in the International Prospective Register of Systematic Reviews (PROSPERO) database (CRD42022340699).in vitro models, published in Portuguese, English or Spanish, were included in the study.The search was applied to the MEDLINE databases via PubMed, Scientific Electronic Library Online (SciELO), Latin American and Caribbean Health Sciences Literature (LILACS), Virtual Health Library (BVS) and Google Scholar. The descriptors were “thyroid cancer” or “thyroid cancer – papillary”, “TPC-1” and “flavonoids” or “polyphenols”. All original articles that evaluated influences exerted by flavonoids on the TPC-1 cell line using No time limit was used for the publications included, as it is a recent topic in literature. Review articles, clinical studies, studies that did not evaluate the effect of flavonoids specifically in TPC-1 cell line, and, gray literature , were excluded.After the initial search for descriptors in databases, the first analysis was performed to exclude duplicate studies. The remaining article contents were verified by title and abstract. This analysis determined the relevance of publications, and those that did not fit the selection criteria were also excluded.The remaining articles were fully analyzed for the elaboration of this systematic review. After the complete reading of the texts, if any study still did not fit the selection criteria, it was also excluded, specifying the reason for the exclusion.The manuscripts selected to compose this review had their data extracted into a spreadsheet (Microsoft® Excel version 16.42) based on the following domains: author names, year of publication, type of study, flavonoids used, concentrations of compounds, time of exposure to the compounds, cellular pathways evaluated and the effect of the polyphenols tested on cell lines.The data obtained were presented through a table, as well as descriptive exposition and discussion of the results.in vitro evaluation was performed (n = 9); articles that were not original (n = 14). Thus, the descriptive synthesis of this review consisted of 11 original articles, all with a pre-clinical design using the TPC-1 cell line criterion and different polyphenols , activators or inhibitors of apoptosis  polymerase-PARP), inflammatory cytokines (tumor necrosis factor – TNFα), inductors of cell growth  and signaling pathways  , cell cycle regulators  .The main results from the selected articles are demonstrated in Thyroid cancer is considered the most common endocrine neoplasm in the world. According to the Global Cancer Observatory, its incidence in 2020 was 6.6 per million individuals, being the fifth most common malignancy diagnosed in women. Papillary thyroid carcinoma represents about 90% of all thyroid cancers . StudiesThyroid cancer is divided into subtypes according to the degrees of cell differentiation and their histological and pathological features, which have strong influence in disease progression and treatment strategies. Among the thyroid cancer subtypes, the well-differentiated carcinomas are the most frequent, such as papillary and follicular thyroid cancer. The other tumors correspond to medullary and anaplastic carcinomas that tend to be more aggressive and less responsive to treatment . The aveSeveral aspects can increase the risk of developing this pathology, including a history of radiation exposure, a family history of thyroid cancer in a first-degree relative and some genetic conditions, such as multiple endocrine neoplasia syndrome. The main treatment for thyroid cancer is surgery , and other postoperative interventions may be needed according to tumor characteristics, such as radioactive iodine therapy .In 2017, the US Preventive Services Task Force published a review against screening thyroid nodules in an asymptomatic general adult population, for it ended up causing greater harm compared to the expected benefits, mainly due to unnecessary surgery and postoperative complications (Papillary thyroid carcinoma (PTC) is the most common histological subtype, accounting for 90% of cases. It develops in follicular cells and has a benign behavior . Most caIn recent years, protein tyrosine kinase inhibitors have emerged as an alternative for treatment in refractory cases of PTC. They target intracellular tyrosine kinase (TK), a protein that catalyzes protein substrates and modulates enzymatic activity, positively interfering in various mechanisms of tumor growth and spread, such as angiogenesis, lymphangiogenesis and proliferation of cancer cells. Different TK inhibitors, such as Subnitib®, Sorafenib® and Vandetanib®, act on several TKs, inhibiting target cells in a non-selective manner. For this reason, severe side effects may occur due to toxicity associated with the difficulty in selecting specific targets and to the use of high doses needed because of the possibility of developing drug resistance ,27.Flavonoid is the nomenclature used to define a class of polyphenolic substances of plant origin . The basIn this context, there are divergences in the literature below regarding the dosage of these compounds in daily diet, since this value is influenced by factors such as: age and sex of the population, method of analysis for absorption in each individual and even annual season and soil characteristics, since they cause differences in the quality of flavonoid substances. Some studies have shown considerable differences in the amount of flavonoid intake per day, which values can vary from 23.0 mg to 1.0 g or 1.1 g ,31.It is also worth highlighting the classification of these substances, since structural differences at the molecular level can promote different biological effects . RegardiAmong the biological effects mentioned – one of great value, especially regarding cancer therapy – is the antineoplastic potential of flavonoids due to their antiproliferative activity. In this sense, many studies reveal the inhibition of cell transformation and proliferation with the administration of flavonoid substances. A North American study describes the influence of some different classes of polyphenols as inhibitors of neoplastic transformation, highlighting the diverse action mechanisms of these compounds used individually or combined . FurtherRegarding flavonoids and the concentrations used in the studies, genistein and quercetin were administered by Kang and cols. (2011) in a 72-hour exposition with concentrations of 10, 50 and 100 μM/mL. Both compounds promoted inhibition of cell growth in a dose-dependent mode in the MTT -2,5-diphenyl-2H-tetrazolium bromide) assay. In the same study, exposition of resveratrol in a concentration of 50 μM/mL for up to 120 hours showed an inhibition of cell growth  at a concentration of up to 50 μM/mL in a 48 hours exposition, and presented different results according to the method of analysis applied. The analysis from the MTT assay revealed a decrease in cell viability in a dose dependent mode; in the flow cytometry method, cell cycle arrest and increase of apoptotic cells was observed; in the Colony formation, a colony formation decrease was found. Western blotting analysis showed an increase of caspase-3, −8 and bax expression, and a decrease of bcl-xl and mcl-1 expression .P21, P53) and apoptosis regulators  were down-regulated (Perna and cols. (2018) evaluated curcumin with a concentration of 25 μM/mL in a 48-hour exposition, showing different cellular responses according to the method of analysis. In the Immunoblotting method, three results were obtained: degraded nrf2, reduced VEGF expression and up-regulated TNFα in 2 of the 3 complexes tested. In the analysis from the MTT assay, there was a reduced cell viability, and in the Western blotting analysis, both cell cycle regulators  in a 48-hour exposition with a concentration of 25 μM/mL. The MTT assay revealed cell viability reduction, and immunofluorescence analysis demonstrated damage to the cell nucleus. There was a down regulation of cell cycle regulators  and apoptosis regulators , verified by Western blotting, similar to that observed by Perna and cols. (2018)  in a 48-.Zhou and cols. (2019) used narigin at concentrations of 6, 12 or 25 μM/mL. In the MTT assay, cell proliferation inhibition was observed, and flow cytometry showed an induction in cell apoptosis. In the Western blotting analysis, there was an enhancement of caspase 3 and bax expression, reduction of cyclin D1, c-Myc, survivin, and bcl-2 expression, as well as the suppression of PI3K/AKT pathway activation (Using extract from Annurca Flesh Apple (AFPE), Orlandella and cols. (2022) applied it in a 24 hours exposition with a concentration of up to 250 μM/mL, resulting in a reduction of cell viability and promotion of cell cycle arrest analyzed by flow cytometry. Another concentration of AFPE (500 μM/mL) in a 20 hours exposition was evaluated by the DCFDA/H2DCFDA – Cellular ROS Assay Kit, obtaining a reduction of cell death induced by oxidative stress, contrasting with the findings described so far .Epigallocatechin-3-gallate (EGCG) showed different cellular responses when used in different concentrations in the study by Wu and cols. (2019). Considering 24 hours of exposure with doses between 10-200 μM/mL, it inhibits cell proliferation and cell viability when analyzed by EdU and MTS assays methods, and causes cell cycle arrest as shown in flow cytometry. In the same article, concentration up to 25 μM/mL, evaluated by TUNEL staining and Western blotting, revealed an increase of the apoptotic index. Concentrations up to 50-200 μM/mL, using Western blotting method, showed an increase of the bax/bcl-2 ratio and the expression levels of cleaved caspase-3 and cleaved PARP. Furthermore, Western blotting was used to analyze exposure with 50-200 μM/mL EGCG, which revealed a gradually decrease of p-EGFR, RAS, p-RAF, p-MEK1/2 and p-ERK1/2 .Prunella vulgaris (PV) was used, a compound with rutin, hyperoside and other flavonoids isolated and identified from PV . At concentrations of 0, 5, 10, 20 and 30% for 12, 24 and 36 hours, based on a CCK-8 assay, cell proliferation inhibition was observed. When used 50% PV for 24 hours, the Hoechst 33342 staining method revealed alteration of cellular morphology and cell nuclei, DNA extraction and fragmentation assay showed induction of cell apoptosis and RT-qPCR indicated decrease of bcl-2 expression and increase of bax and caspase-3 expression (In the study by Yin and cols. (2017) Fisetin was used in the research conducted by Liang and cols. (2020) at concentrations from 1 μM/mL to 30 μM/mL for 24 hours, promoting a cytotoxic effect evidenced by the MTT assay. At 15 μM/mL dose, through the morphological examination in a fluorescence microscope, induction of cell apoptosis without compromising cellular membrane was visualized. In the concentration of 20 μM/mL, still using the same analysis, an elevated quantity of late apoptotic cells was observed. The same dose revealed ROS enhancement, shown by DCFH-DA dye method, and reduction of the mitochondrial membrane potential was detected by spectrofluorimetry. The concentration of 15-20 μM/mL was analyzed by flow cytometry which showed an alteration of cell cycle progression in a dose dependent mode. The Western blotting method revealed a down regulation of the cyclin D1 expression; up regulation of STAT3 and decreased expression of JAK-1. ELISA analysis showed an elevated protein expression of cleaved caspase-3, and −9 in a dose dependent mode .Silibinin was applied by Oh and cols. (2013) with a 100 μM/mL dose for 24 hours, which revealed a decrease in cell viability with the quick cell proliferation assay kit II . XanthohIn summary, all polyphenol substances studied had a molecular influence in the TPC-1 cell lineage, mostly in a dose and time dependent manner. Cellular proliferation was inhibited in all studies analyzed, most of them showed by the MTT assay ,12,14,36In vitro studies obtained results by using concentrations above usual dietary consumption, and tested the toxicity in control cells, without evidence of cell damage. However, this result is limited to cellular analysis, and this evidence should be used for studies in animal models, aiming to know the real safety and efficacy of these substances in this scenario. According to this evidence, some studies have already tested these polyphenolic compounds associated with antineoplastic drugs and the results, so far, are promising. For example, cisplatin administered with curcumin in mice potentiates the cytotoxicity via mediating cell death and cycle arrest, enhancing drug sensitivity and preventing resistance, in addition to reduced cisplatin-mediated side effects (Therefore, it is possible to consider that many polyphenolic compounds have antineoplastic properties specifically in TPC-1 cell lineage.  effects . Specifi effects . Another effects .in vitro studies using high doses of phytochemicals. Their usual consumption was not able to demonstrate protection or risk of inducing neoplasms, whether thyroid or other primary foci. In addition to the amount of exposure to the phytochemical, one must consider the factors that influence the extraction of the active ingredient, which vary according to location, sun exposure, soil characteristics, among other factors. Therefore, to extrapolate these findings to all substances, it would be necessary to rigidly standardize the substances and adequately measure it in individuals who, in addition to the confounding factors related to the phytochemical, also suffer influences in the individual's organism, where absorption can be influenced by intestinal flora, interactions with other medications and other possible influencers. Therefore, an animal model with greater possibility of controlling individual factors and a standardization of the substance would be necessary to carry out more in-depth studies on this topic (Although relevant, these findings are reflected in is topic ,42.In conclusion, there is evidence that polyphenolic compounds have antineoplastic properties on a cellular level, through mechanisms such as inhibiting cell growth, increasing expression of apoptotic proteins, stimulating down regulation of cell cycle regulators and causing DNA damage, but, so far, in doses above those usually obtained through dietary consumption. There is also no evidence of toxicity considering cellular models. These findings reveal a possible therapeutic potential of polyphenols due to their direct effects on neoplastic cells and their actions combined with antineoplastic drugs. However, these results deserve further studies, advancing to trials in animal models and in association with other substances such as tyrosine kinase inhibitors, and, seeking alternatives for the treatment of refractory cases, which are extremely limited in current treatments.


Background: Melanoma represents the deadliest skin cancer due to its cell plasticity which results in high metastatic potential and chemoresistance. Melanomas frequently develop resistance to targeted therapy; therefore, new combination therapy strategies are required. Non-canonical signaling interactions between HH-GLI and RAS/RAF/ERK signaling were identified as one of the drivers of melanoma pathogenesis. Therefore, we decided to investigate the importance of these non-canonical interactions in chemoresistance, and examine the potential for HH-GLI and RAS/RAF/ERK combined therapy. Methods: We established two melanoma cell lines resistant to the GLI inhibitor, GANT-61, and characterized their response to other HH-GLI and RAS/RAF/ERK inhibitors. Results: We successfully established two melanoma cell lines resistant to GANT-61. Both cell lines showed HH-GLI signaling downregulation and increased invasive cell properties like migration potential, colony forming capacity, and EMT. However, they differed in MAPK signaling activity, cell cycle regulation, and primary cilia formation, suggesting different potential mechanisms responsible for resistance occurrence. Conclusions: Our study provides the first ever insights into cell lines resistant to GANT-61 and shows potential mechanisms connected to HH-GLI and MAPK signaling which may represent new hot spots for noncanonical signaling interactions. V600E or NRASQ61R mutations which lead to constitutive activation of the RAS/RAF/ERK signaling and, consequently, the promotion of cell proliferation, survival, invasion, and angiogenesis . T. T15]. TGLI1, GLI2, GLI3, PTCH1, SHH, SUFU, SMO and GSK3B , cell proliferation , and stemness   C. Both Gpression A. Howevemponents B. Unexpeoperties C. The Me R cells C.DUSP4, DUSP7, SPRY2, and SPRY4  levels. Both resistant cell lines exhibited slightly downregulated levels of phospho-JNK. Finally, we used a flow cytometry approach to investigate MAPK/PI3K signaling activity modulation by acute treatment with an MEK inhibitor (U0126) or GANT-61 in parental and resistant cells. The kit detects phospho-ERK1/2 and phospho-AKT positive populations and, therefore, identifies the activation of the MAPK, the PI3K, or both signaling pathways. For that reason, flow cytometry enables us to quantify trends that we previously observed with western blot analysis—namely, ERK signaling hyperactivation. The Mel 224 and Mel 224 R cell lines showed similar MAPK/PI3K activity profiles in basal conditions  (light blue column segments). Interestingly, acute treatment with 10 mM and 20 mM GANT-61 increased the MAPK positive cell population in Mel 224 R compared to the Mel 224 cells (p = 0.001). The CHL-1 and CHL-1 R cell lines showed a similar response to U0126 and GANT-61 but not statistically significant: as we described for Mel 224 R, the CHL-1 R cells appeared more sensitive to U0126 treatment than CHL-1, and showed an increase in MAPK/PI3K negative cell population.Given the known crosstalk between the HH-GLI and MAPK signaling and their involvement in melanoma development, we also examined the expression and activation status of ERK1/2, JNK, and p38 MAPKs at the protein level. The Mel 224 R cells showed a dramatic increase in the level of phospho-ERK and reduction of phospho-p38, while the CHL-1 R cells had an opposite pattern: increased phospho-p38 level and a reduction in ERK signaling D. The opnd SPRY4 E. These p = 0.0095), CYC , and LiCl —while, in the CHL-1 R cells, only LiCl  showed a significant dose-dependent decrease in colony formation capacity. Once we successfully validated our resistant cell lines, we examined their response to other HH-GLI and RAS/RAF/MAPK inhibitors in terms of proliferation and invasive cell properties like colony formation capacity and migration. To determine the response to other inhibitors, we performed MTT viability assays as previously described. Both Mel 224 and Mel 224 R cell lines showed a dose-dependent reduction of viability in response to arsenic trioxide (ATO), a non-selective GLI protein inhibitor ,17; howep = 0.0003 for Mel 224 R and 24 h p = 0.0027 for CHL-1 R) (p = 0.0134) compared to CHL-1. In comparison to untreated conditions, LiCl was the most effective in attenuating migration potential in both CHL-1 cell lines (24 h p = 0.0179 for CHL-1 and 24 h p = 0.0001 for CHL-1 R).Migration analysis showed that both resistant cell lines had a higher migration potential under basal conditions in comparison to the parental cell lines (24 h CHL-1 R) C. Overalp < 0.0001), while this effect was not observed in the parental cell line; however, a significant increase in the sub-G1 population which represents apoptotic cells was detected instead (p < 0.0001), (p < 0.0001), while G0/G1 accumulation was observed in the resistant cell line (p = 0.0002). In all samples, regardless of the conditions, we could detect a multinucleated cell population, which can be identified as a signal after G2/M cell population during cell cycle analysis . In the CHL-1 cell line, cell cycle profiles differed between the parental and resistant cells. In untreated conditions, the CHL-1 R cell line had a higher percentage of G0/G1 cell population in comparison to CHL-1 (p = 0.0001). Contrary to the Mel 224 cell lines, DOXO treatment caused the expected G2/M accumulation only in the CHL-1 cell line (p < 0.0001), while the CHL-1 R cell line had a higher sub-G1 population percentage (p < 0.0001), indicating apoptosis. With GANT-61 treatment, we have observed the same effect—G0/G1 accumulation in CHL-1 (p = 0.0001) and increased sub-G1 population in CHL-1 R cell line (p = 0.0001) , B 21]. W. Wp < 0.analysis . Multinu 0.0001) B. seq and ChIP-seq approach. RAB34, as a novel GLI2 transcriptional target, is a crucial regulator of primary cilia formation [p = 0.0308). The finding that the cells that form the primary cilia represent a small per cent of the total population is not a surprising observation since it is known that melanoma cells often show primary cilia loss, which can be connected with increased oncogenic properties [In one of our previous studies, we identified novel GLI transcriptional targets using a combined RNA-ormation . Since coperties ,24. TherThe RAS/RAF/MAPK and HH-GLI signaling pathways are known to interact in melanoma and to promote tumorigenic properties like cell proliferation, survival, and invasiveness . ChemoreWT cell line was more sensitive to long-term GANT-61 treatment than the Mel 224 NRASQ61R cell line, as it was harder to establish permanent resistance. These results are consistent with studies showing that the NRAS mutated melanoma is more aggressive, and associated with elevated mitotic activity and higher metastatic properties when compared to the NRAS wild-type melanoma [V600E mutated melanoma lines, which will be investigated in the future. Our established cell lines displayed a great number of molecular and morphological changes, with some commonalities, but mainly showing different mechanisms underlying GANT-61 resistance. One of the observations in both resistant cell lines, which was distinctly noticeable, was the change in cell morphology. In comparison to the parental cell lines, the resistant cell lines had a spindle-like shape and were bigger in size. Our hypothesis that these changes might be associated with EMT was confirmed with the detection of downregulated gene levels of the epithelial marker CDH1 and upregulated levels of the mesenchymal marker VIM in the resistant cell lines. It is known that cells undergoing EMT have increased invasive properties by producing more matrix metalloproteinases (MMPs) [MMP2 gene levels in both resistant cell lines. Besides being involved in cell invasion, EMT-related transcription factors can trigger the expression of stemness factors [OCT4, NANOG, and SOX2 [NANOG and SOX2 levels, while the CHL-1 R cell line showed upregulation of OCT4, suggesting increased stemness in both cell lines through different mechanisms. In this study, we present for the first time an in-depth characterization of two melanoma cell lines resistant to GANT-61, a specific GLI protein antagonist. The CHL-1 NRASmelanoma . Howevers (MMPs) . In our  factors . Since cand SOX2 . The MelOn the protein and mRNA levels, both resistant cell lines exhibited downregulation of the majority of the analyzed HH-GLI signaling components. Protein levels of all specific HH-GLI signaling components  were downregulated in the resistant cell lines. In the case of GLI1 protein, we identified two isoforms, the 160 kDa full length protein isoform (GLI1FL) and a ~130 kDa isoform, possibly the GLI1ΔN which is lacking the N-terminal SUFU binding domain . TherefoIt is known that tumors originate from multiple clones of malignant cells with different properties, which can be selected due to applied therapy . TherefoSPRY2, SPRY4, DUSP4, and DUSP7. Consistent with our previous observations, SPRY4, DUSP4, and DUSP7 were downregulated in Mel 224 R, while SPRY2, SPRY4, and DUSP4 were upregulated in the CHL-1 R cell line. Balko et al. demonstrated that MAPK activation upon DUSP4 loss promotes cancer stem cell-like properties in basal-like breast cancer cells [V600E and NRASQ61 oncogene expression [Interestingly, we observed that different MAPK signaling pathways were hyperactivated in the resistant cell lines. MAPK signaling is composed of three main cascade modules, ERK1/2, p38, and JNK, each of which has its own substrates and specific biological functions . The ERKer cells . Yao et pression ,42. To characterize more thoroughly our established cell lines, we examined their response to other HH-GLI and RAS inhibitors. HH-GLI inhibitors can target upstream and downstream components of the signaling cascade. Cyclopamine, vismodegib, and sonidegib bind to SMO on the cell membrane and inhibit downstream signaling transduction ,44. LithMMP2 gene levels in both resistant cell lines, we wanted to examine if they exhibit elevated invasive traits by analyzing cell migration and colony formation capacity. In comparison to the parental cell lines, Mel 224 R and CHL-1 R showed a higher migratory and colony formation capacity upon treatment with HH-GLI inhibitors, GANT-61, ATO, CYC, and LiCl. A study of Yang et al. showed that RAS/RAF/ERK signaling can mediate MMPs’ activity which influences cell migration and invasion [Since we have previously detected upregulated invasion . Guided invasion ,54. seq and ChIP-seq [RAB34 gene, a novel GLI2 transcriptional target, is required for fusion of preciliary vesicles and, consequently, for normal primary cilia formation. HH-GLI signaling is highly dependent on proper cilia formation and it has been shown that RAB34 activity is crucial for canonical HH-GLI signaling [In a previous study, we identified numerous novel GLI transcriptional targets by combining RNA-ChIP-seq . The RABignaling . Since wignaling . A studyignaling ,57. Primignaling ,59. We dHere we report, for the first time, a comprehensive characterization of melanoma cell lines of different genetic backgrounds resistant to GANT-61. Although we established two cell lines resistant to the same drug, potential mechanisms involved in resistance occurrence differ between them. However, there are shared traits among them which need to be investigated further. Both cell lines exhibited HH-GLI signaling downregulation and switching to different modules of MAPK signaling, cancer stem-like properties and elevated invasive characteristics visible through increased migration, colony formation capacity, and EMT. The established cell lines did not show permanent cross-resistance to other tested HH-GLI and RAS inhibitors. Our study provides new insights into the HH-GLI and RAS/RAF/ERK interplay associated with ciliogenesis regulation via RAB34.


Enterococcus faecalis KUMS-T48, isolated from a kind of Iranian traditional dairy product (Tarkhineh), was assessed for its anti-pathogenic, anti-inflammatory and anti-proliferative properties against HT-29 and AGS cancer cell lines. This strain showed strong effects on Bacillus subtilis and Listeria monocytogenes and moderate effect on Yersinia enterocolitica, while indicated weak effect on Klebsiella pneumoniae and Escherichia coli. Also, neutralizing the cell-free supernatant and treating it with catalase and proteinase K enzymes reduced the antibacterial effects. Similar to Taxol, the cell-free supernatant of E. faecalis KUMS-T48 inhibited the in vitro proliferation of both cancer cells in a dose-dependent manner, but unlike Taxol, they had no activity against normal cell line (FHs-74). Pronase-treatment of the CFS of E. faecalis KUMS-T48 abrogated its anti-proliferative capacity, thereby showing the proteinaceous nature of the cell-free supernatant. Further, induction of apoptosis-based cytotoxic mechanism by E. faecalis KUMS-T48 cell-free supernatant is related to anti-apoptotic genes ErbB-2 and ErbB-3, which is different from Taxol’s apoptosis induction (intrinsic mitochondria apoptosis pathway). Also, as evidenced by a decline in interleukin 1β inflammation-promoting gene expression and a rise in the anti-inflammatory interleukin-10 gene expression in the HT-29 cell line, probiotic E. faecalis KUMS-T48 cell-free supernatant demonstrated a significant anti-inflammatory impact.Potential probiotic  Tarkhineh is a kind of traditional fermented food that is popular in the western provinces of Iran as well as in many different countries such as Armenia, Iraq, Turkey, Greece, and Bulgaria3. Iranian Tarkhineh, which is rich in probiotic lactic acid bacteria (LABs), is made up of wheat or barley grouts that is cooked for hours in sour milk, and the pieces of this paste-like mixture are balled and dried after adding local vegetables such as turnips and oregano4.Recent discoveries on the benefits of food bacterial populations on human health motivate further interest in bacterial communities and their functions5. Several studies have revealed that the majority of probiotic bacteria belong to the lactic acid bacteria group6. LABs exhibit fermentation activities and have been used in food preservation for thousands of years. The LAB groups include the genera such as Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, Oenococcus, Streptococcus, and Pediococcus8.Probiotic strains with health-promoting properties are known as bio-therapeutic agentsEnterococcus has the third highest number of species in the LAB group following Lactobacillus and Streptococcus genera9. They are catalase-negative, Gram-positive, and non-spore-forming bacteria found in a variety of fermented foods10. E. faecium and E. faecalis are two predominant species among Enterococcus species11. The application of probiotic Enterococcus species has been recently augmented as a consequence of their affirmative effects on human health12.13. However, anti-carcinogenic activity is the most interesting property that has been linked to probiotics15. In this regard, different studies have also been conducted on the anti-proliferative effects of food-derived enterococci on various cancer cell lines18. Apoptosis induction and down-regulation of genes involved in cancer cell proliferation are two effective mechanisms of probiotic cell-free supernatant in protective effects against many cancers20. This study aimed to assess the anti-carcinogenic, anti-inflammatory, and anti-pathogenic activity of probiotic Enterococcus faecalis KUMS-T48 isolated from Tarkhineh.Probiotics, as health-promoting microorganisms, show different therapeutic properties such as anti-pathogenic and cholesterol-lowering activitiesEnterococcus faecalis KUMS-T48, which was previously isolated from Tarkhineh, and its molecular identification and potential probiotic properties were also evaluated7. The de Man Rogosa Sharpe (MRS) medium was used for the activation and growth of this strain.The present study was conducted on 21 was used to determine the antagonistic effect of E. faecalis KUMS-T48 cell-free supernatant on five indicator pathogens including Listeria monocytogenes (PTCC 1163), Escherichia coli (PTCC 1276), Klebsiella pneumoniae , Yersinia enterocolitica (ATCC 2715), and Bacillus subtilis . For this, 50 μL of the filtrate (through 0.2 µm filter) of an overnight culture of E. faecalis KUMS-T48 in MRS broth at 37 °C was added to 7 mm diameter wells on MRS agar plates , which were previously surface cultured with indicator pathogens and incubated overnight at 37 °C. After overnight incubation of plates at 37 °C, the clear zones around of each well were measured and considered as positive antibacterial activity. According to the diameter of the inhibition zone (average of two perpendicular diameters), the anti-pathogenic activity was divided into strong (diameter ≥ 20 mm), moderate (10 mm < diameter < 20 mm), and weak (diameter ≤ 10 mm). In order to determine the primary ingredient involved in the antagonistic properties of the cell-free supernatant, the neutralized form (adjusted to pH 7.2 by adding 1 M NaOH) and treated forms of the cell-free supernatant with catalase and proteinase K enzymes were also subjected to antibacterial tests.The modified well diffusion agar method previously described by Nami et al.Enterococcus strain was cultured on MRS broth and incubated at 37 °C for 24 h. By measuring the bacterial optical density (OD), 1 × 106 CFU mL−1 (standard number of viable probiotic cells) was chosen as the ready-to-use cell culture. The active supernatants were separated via centrifugation at 4000× g for 30 min at 4 °C of cell cultures. Then, the supernatant was filtered through a 0.22 µm pore-size filter after neutralizing by adding 1 M NaOH22.For this purpose, the selected 23. All cell lines were cultured in 96-well cell culture plates containing Dulbeccos Modified Eagles Medium , where the media were supplemented with 100 IU mL−1 penicillin, 10 µg mL−1 streptomycin, and 10% (v/v) fetal bovine serum at similar incubation conditions for all cells . All cultured cells were subsequently treated with 12 concentration rates (5–60 µg mL−1) of resulted CFS to screen the effective and suitable concentration for continuing the experiments at three-time points .The human gastric cancer cell line (AGS), human colon cancer cell line (HT-29), and human normal cell line (FHs-74), which were provided by the Tehran Institute of Pasteur , were employed in the bacteria-secreted metabolite cytotoxicity assays24. For this, the cancer cell lines and normal cell lines were plated in the 96-well microplate. Each well was seeded with 1.2 × 104 cells in 150 µl of growth medium. After 24 h post-seeding (40% to 60% confluency), 50 µL (30% (v/v)) of the CFS were administered to each well to achieve a total volume of 200 µL. All treated cells were then incubated for 12, 24, and 48 h. After incubation, 50 µl of MTT solution (5 mg ml−1 in PBS) was administered to each microplate well. Then, the plates were again incubated for 4 h in 5% CO2 at 37 °C in dark conditions. The formazan crystals created by using MTT-exposed live cells were dissolved by adding 200 µl of dimethyl sulfoxide (DMSO) and 25 µL of Sorenson’s glycine buffer  into each well and were incubated at 37 °C while being gently shaken for 20 min. Adsorption of the dissolved formazan crystals was measured using a μQuant ELISA Reader  at 570 nm. The cytotoxic assessments were continued using Taxol-treated groups as a positive control, and untreated cell lines as a negative control. For further comparison, the results were compared with the cell-free supernatant of the reference E. faecalis strain .The MTT -2,5-diphenyltetrazolium bromide) assay was used to detect viable cells25. Briefly, all treated/untreated cell lines were cultured on sterile cover slip sets at the bottom of each 6-well cell culture plate (cell density 120 × 104/each well). At the end of each time point , the cells were fixed by 4% paraformaldehyde (PFA) for 5 min and were permeabilized by 0.1% Triton X-100 for 5 min. Then, 50 µl of diluted 4′,6-diamidino-2-phenyl indole (DAPI) was diluted 1:2000 with its buffer  and added to each well. After 5 min incubation at room temperature, the cells were washed with sterile PBS (pH 7.2). The stained cells on cover slips were reversely placed on the slides and then were analyzed using a fluorescent microscope  equipped with a U-MWU2 fluorescence filter .This test was carried out using the previously described methodReal-time PCR was used to examine seven apoptotic expression genes, including ErbB-2, ErbB-3, BAX, BCL-2, BCL-XL, CASP-8, and CASP-9 Table . AGS canhttp://www.premierbiosoft.com) using public gene bank sequences and are listed in Table Primers were built using Beacon Designer 5.01  was added to the CFS at a concentration of 1 mg mLEnterococcus faecalis KUMS-T48 strain on the expression of interleukin 10 (IL-10) and interleukin 1β (IL- 1β) inflammation-related genes in HT-29 cancer cell line, cells were treated with different concentrations of CFS  in 25 cm flasks as well as one flask as a control in RPMI-1640 culture medium. After 48 h, the cells were separated from the bottom of the flask.To investigate the effect of probiotic RNA extraction was done using high pure kits of Roche Company based on the company's protocol, and then the concentration of extracted RNA was determined using a nanodrop spectrophotometer. Then, due to the instability and single-stranded nature of RNA, cDNA synthesis was performed using a Fermentase kit, and Real-Time PCR technique with primers listed in Table P < 0.05; **P < 0.01) was considered statistically significant. Data were presented as the mean ± standard deviation of three measurements.All data were analyzed using one-way ANOVA and Duncan statistical tests using SPSS 19.0 software. A p-value  after 48 h. The cytotoxic time and dose-dependent curves were used to calculate the 50% inhibitory concentration (IC50) of strain cell-free supernatant as an indication of anti-proliferative activity. After 12 h of incubation, the IC50 values of E. faecalis KUMS-T48 secretions on HT-29 and AGS cancer cell lines were respectively 39 and 32 µg mL−1, which reached 21 and 17 µg mL−1 at the end of the incubation period (48 h), respectively  on AGS, HT-29, and FHs-74 cell lines for 48 h have been illustrated in Fig. E. faecalis PTCC 1774) as control, cell viability decreased significantly (P < 0.01) in both human cancer cell lines treated by E. faecalis KUMS-T48 cell-free supernatant, similar to the effect of Taxol, indicating that the cell-free supernatant had the same anticancer effect , the cell viability of these cells treated with E. faecalis KUMS-T48 cell-free supernatant was found to be 94% at the same time .The cytotoxic effects of 50 µg mL− on AGS, 50 µg mL− on AGS, 50 µg mL− on AGS, P < 0.05) in both cancer cell lines after treatment with E. faecalis KUMS-T48 cell-free supernatant (50 µg mL−1) for 24 h. The fluorescent photomicrographs of DAPI-stained AGS cancer cells are shown in Fig. The results revealed that the numbers of apoptotic cells with the fragmented and condensed nucleus were significantly higher than normal cells  and ErbB-2, ErbB-3 (anti-apoptotic genes) was significantly different in E. faecalis KUMS-T48 and Taxol treated groups  on both HT-29 and AGS cell lines same as Taxol, compared to control sample. The cell viability percentage of treated cells with E. faecalis KUMS-T48 cell-free supernatant and Taxol during incubation time (48 h) were respectively detected 18% and 13% for HT-29 and 21% and 20% for AGS cell lines. However, compared to those on un-treated cells, secretions had very little effect on cancer cells after treatment with pronase, highlighting the critical role of secreted proteins in cytotoxic effects  up to a concentration of 150 µg mL−1 in a dose-dependent manner. Conversely, the expression of the anti-inflammatory IL-10 gene treated with E. faecalis KUMS-T48 increased significantly with increasing the concentration of supernatant up to 200 µg mL−1 (P < 0.01).In this research, the changes in interleukin 10 and interleukin 1β genes expression in cancerous cell lines was investigated. The results of this assay on cancerous cells treated with the CFS of probiotic 28. The findings showed that the antagonistic activity of E. faecalis KUMS-T48 cell-free supernatant was retained after neutralizing CFS and treatment with catalase enzyme, while its activity was completely lost by subjecting it to proteinase K enzyme. So it can be concluded that the antagonistic effect may not be related to acid or H2O2 production and could be attributed to bacteriocins. These findings are consistent with the results reported by others29. Similar to our results, the antagonistic activities of various Enterococcus strains against the diversity of pathogenic bacteria have been previously reported30. The effects of E. faecalis KUMS-T48 cell-free supernatant on Gram-positive bacteria were significantly higher than that of Gram-negative bacteria, which may be related to the outer membrane of Gram-negative bacteria31.In this work, traditional dairy products in the west of Iran were preliminarily screened because different preparation and processing methods can be possibly used as valuable tools to introduce novel and promising probiotic bacteria33. Most studies on the role of probiotic bacteria in cancer prevention have focused on their anti-colorectal cancer effects35 whereas, our results indicate that the cell-free supernatant of E. faecalis KUMS-T48 displayed cytotoxic effects on other cancer cell lines, similar to Taxol. Compared to other cancer cells such as hematological cell lines, HT-29 and AGS , are less sensitive to cytotoxic agents and drugs36. Therefore, the effective agents on them may have better conditions to be used as anticancer agents.Probiotics, as health-promoting microorganisms, exhibit different therapeutic activities. However, anti-carcinogenic activity is the most interesting property that has been linked to probiotics37. Hence, finding safe and inexpensive alternatives such as probiotic metabolites is important. Anti-cancer agents can generally show cytotoxic effects on some sensitive cells and tissues, such as the bone marrow, stem cells, and hair follicle cells39. In this study, E. faecalis KUMS-T48 cell-free supernatant did not show any significant cytotoxic effects on rapidly dividing normal cell lines (FHs-74), so they could be prescribed more safely.As a conventional anticancer drug, Taxol is relatively expensive, and its original source (Pacific yew tree) is limited. In addition, this drug has a cytotoxic effect on non-cancerous cells40. This process explains the cytotoxic effects of pronase-untreated secretions of E. faecalis KUMS-T48 on the AGS cancer cell line.In the present study, the anti-proliferative effect of pronase-treated/untreated secretions was investigated on the cell lines. Our results show that active proteins serve a key function in the cytotoxicity of the cell-free supernatant. Proteins suppress the cancer cell lines by binding to pro-carcinogenic compounds, carcinogenic fecal enzymes, or mutagenic substances41, prostate42 or colon20 cancer cell lines. Methods utilized to assess apoptosis ranged from classical biochemistry and simple light microscopy to the application of sophisticated instruments, such as flow cytometer43. However, cellular morphological characteristics should be considered when determining the mode of cell death. Therefore, the DAPI staining method flowing fluorescent microscopy is still considered the gold standard for analyzing apoptosis44. Various forms of apoptotic bodies were observed in cancer cells treated with E. faecalis KUMS-T48 cell-free supernatant. These changes in cell morphology during apoptosis have also been reported by other researchers45. The apoptotic cells can be distinguished from necrotic and viable cells based on nuclear morphology, such as nucleus fragmentation and chromatin condensation46. The morphological apoptotic results of this work indicate that apoptosis is the main cytotoxic mechanism for the cell-free supernatant of E. faecalis KUMS-T48 on cancer cell lines and the necrotic bodies were rarely observed.The association between the anticancer activity of therapeutic agents and apoptosis has been shown in different cell lines, such as human cervical47. We also showed that the expression of intrinsic apoptosis blocker genes (BCL-2 and BCL-XL), CASP-8 gene (starter gene in TNF-α apoptosis pathway), and BAX  were significantly down-regulated by E. faecalis KUMS-T48 compared to the untreated control group. The cell-free supernatant from E. faecalis KUMS-T48 increased the expression of ErbB-2 and ErbB-3 genes, while Taxol increased the expression of CASP-9, suggesting alternative apoptosis-inducing mechanisms. According to these results, the activation of apoptosis by E. faecalis KUMS-T48 The cell-free supernatant from E. faecalis KUMS-T48 increased the expression of ErbB-2 and ErbB-3 genes, while Taxol increased the expression of CASP-9, suggesting alternative apoptosis-inducing mechanisms is associated with the down-regulation of anti-apoptotic genes (BCL-2 and BCL-XL), but differs from Taxol's apoptosis induction  route. The molecular mechanisms of the pro-apoptotic effects of human-derived Lactobacillus reuteri (ATCC 6475) on myeloid leukemia-derived cells have previously been investigated, with findings indicating down-regulation of nuclear factor kappa B (NF-kappa B)-dependent gene products that mediate cell survival-related genes (BCL-2 and BCL-XL). Lactobacillus paracasei M5L, according to Hu et al.48, may trigger apoptosis in HT-29 cells through reactive oxygen species production, followed by CRT-accompanied endoplasmic reticulum stress and S phase arrest. The impact of probiotic Bacillus polyfermenticus on the proliferation of human colon cancer cells such as HT-29, DLD-1, and Caco-2 cells has been discovered. It has been observed that the apoptosis-inducing effect of Bacillus polyfermenticus metabolite is connected to the repression of the ErbB-2 and ErbB-3 genes, which resulted in this strain's anti-tumor characteristics49. Furthermore, the anticancer effects of cell-bound exopolysaccharides (cb-EPS) isolated from Lactobacillus acidophilus 606 on HT-29 colon cancer cells have been shown by the expression of BAX gene50.The expression of pro-apoptotic markers (CASP and BAX) and anti-apoptotic proteins (BCL-2 and BCL-XL) is used as an indicator of apoptosis induction in tumor cells51. Probiotic lactobacilli, bifidobacteria, and E. faecium have been linked to anti-inflammatory effects in the past53. In the present study, the cell-free supernatant from E. faecalis KUMS-T48 increased the expression of ErbB-2 and ErbB-3 genes, while Taxol increased the expression of CASP-9, suggesting alternative apoptosis-inducing mechanisms of probiotic E. faecalis KUMS-T48 demonstrated a strong anti-inflammatory impact, as evidenced by a decrease in interleukin 1β gene expression and an increase in the anti-inflammatory interleukin-10 gene expression in the HT-29 cell line. Reducing the release of inflammatory mediators such as IL-1β, or increasing the level of IL-10 as an anti-inflammatory cytokine, are considered in studies investigating anti-inflammatory effects54. IL-1β is involved in inflammation and tumor growth by stimulating angiogenesis in the cancerous cells55. However, IL-10 is a strong inhibitor of the synthesis of pro-inflammatory cytokines such as TNF-α56.Previous studies implies that various bacteria may have potential to be used as anti-inflammatory agentsE. faecalis KUMS-T48 secretions on cancerous HT-29 and AGS cell lines were similar to that of Taxol, which is a conventional anticancer drug. Taxol possesses high cytotoxicity on normal cell lines, but the cell-free supernatant from E. faecalis KUMS-T48 increased the expression of ErbB-2 and ErbB-3 genes, while Taxol increased the expression of CASP-9, suggesting alternative apoptosis-inducing mechanisms of E. faecalis KUMS-T48 did not show any significant cytotoxic effects on rapidly dividing FHs-74 normal cell lines. Furthermore, E. faecalis KUMS-T48 cell-free supernatant showed remarkable antibacterial activity and anti-inflammatory effects. These antimicrobial properties or anti-proliferative effects of the cell-free supernatant that were induced by apoptosis were lost after subjection to proteolytic enzymes. Hence, these secreted proteins have the potential to be introduced as anticancer therapeutics. Of course, they should be comprehensively examined in terms of physicochemical, structural, and functional properties before use.In conclusion, the cytotoxic effects of


AK-HW-90 (2b) as a potent pan-BCR-ABL inhibitor against imatinib-resistant mutants, particularly T315I. A concise array of six compounds 2a–f was designed based on our previously reported benzothiazole lead AKE-5l to improve its BCR-ABLT315I inhibitory activity. Replacing the 6-oxypicolinamide moiety of AKE-5l with o-methoxyphenyl and changing the propyl spacer with phenyl afforded 2a and AK-HW-90 (2b) with IC50 values of 2.0 and 0.65 nM against BCR-ABLT315I, respectively. AK-HW-90 showed superior anticancer potency to imatinib against multiple cancer cells (NCI), including leukaemia K-562. The obtained outcomes offer AK-HW-90 as a promising candidate for the treatment of CML and other types of cancer.The design of kinase inhibitors targeting the oncogenic kinase BCR-ABL constitutes a promising paradigm for treating chronic myeloid leukaemia (CML). Nevertheless, the efficacy of imatinib, the first FDA-approved targeted therapy for CML, is curbed by the emergence of resistance. Herein, we report the identification of the 2-methoxyphenyl ureidobenzothiazole   Chronic myeloid leukaemia (CML) is a myeloproliferative neoplasm characterised by excessive production of immature white blood cells and constitutes approximately 15% of leukaemia cases in adultsImatinib , a diar,T315I inhibitors with improved selectivity/safety have been developed , it was evident that installing 2-methoxyphenyl motif directly at C-6 of benzothiazole is optimal for activity than 6-oxypicolinamide. Therefore, in the current study, we replaced the picolinamide ether of AKE-5l with 2-methoxyphenyl moiety, while conserving the ureidobenzothiazole scaffold benzo[d]thiazol-2-yl)-3-phenylurea derivatives 2a–f. All target compounds were evaluated against both BCR-ABLWT and BCR-ABLT315I, and a representative derivative 2b was further profiled against an array of imatinib-resistant BCR-ABL mutants.In a previous study, our group reported a series of benzothiazoles featuring picolinamide moiety as BCR-ABL inhibitorsAKE-5l  was idenscaffold . In addisupplemental material.The detailed experimental section is included in the d]thiazol-2-amine (1), was obtained via Suzuki coupling of 2-amino-6-bromobenzothiazole with (2-methoxyphenyl)boronic acid using Pd(dppf)Cl2• CH2Cl2 as a catalyst under two different conditions. Initially, the reaction was conducted in 1,4-dioxane:H2O,  at 95 0C for 4 h using potassium carbonate as a base, and afforded compound 1 in 58% yield. Changing the solvent with 1,2-dimethoxyethane (DME):H2O , using sodium bicarbonate and running the reaction at 85 0C for 2 h furnished 1 in higher yield (87%). Treatment of benzothiazol-2-yl amine 1 with 1,1′-carbonyldiimidazole (CDI) in DMF afforded the corresponding isocyanate, which was coupled with the proper aniline at 100 0C to generate the target ureidobenzothiazoles.The synthetic outline for the preparation of the final compounds is illustrated in 2a–f were tested against the native BCR-ABLWT along with its clinically imatinib-resistant T315I mutant at Reaction Biology Corporation 2a–c, with hydrophilic cyclic amine at the 4-position of the terminal phenyl ring, showed excellent inhibitory potency with IC50 values spanning in the range of 0.701–2.16 nM and 0.651–8.11 nM against BCR-ABLWT and BCR-ABLT315I, respectively, being superior to the lead compound AKE-5l . Such biochemical outcomes indicate that replacing the oxypicolinamide moiety of AKE-5l with o-methoxyphenyl, along with changing the propyl spacer into phenyl, resulted in improved BCR-ABL inhibitory activity. The existence of terminal nitrogen with small alkyl substituents, 2a and 2b, was found to be optimal for BCR-ABL inhibition. Compound 2b, with N-ethylpiperazine, exerted the best activity with sub-nanomolar IC50 values of 0.701 and 0.651 nM against BCR-ABLWT and BCR-ABLT315I, respectively. It showed 2–3 folds better activity than its methylpiperazine congener 2a . Replacing the terminal nitrogen of piperazines 2a and 2b with oxygen 2c retained the BCR-ABLWT activity, however, it led to reduction in BCR-ABLT315I inhibition (BCR-ABLT315I IC50 = 8.11 nM). Insertion of m-trifluoromethyl group on the distal phenyl while removal of the hydrophilic cyclic amines 2d resulted in a dramatic drop in potency . Such decline in activity was much more pronounced upon the introduction of an additional trifluoromethyl group at the 5-position, as in 2e . In contrast, incorporation of morpholine methylene at the 4-position of 2d afforded 2f, which restored inhibitory potency with IC50 values of 74.6 and 226 nM against BCR-ABLWT and BCR-ABLT315I, respectively. These findings point out the indispensable role of cyclic amines, particularly piperazine, in achieving sound BCR-ABL inhibition for this set of ureidobenzothiazoles. This importance might be attributed to the hydrophilic nature of cyclic amines, which offers a favourable orientation towards the solvent exposure region of BCR-ABL. Such a hypothesis could be emphasised by noticing the detrimental impact of the existence of m-trifluoromethyl group (2d and 2e) on the distal phenyl ring.All final compounds 2b was further profiled against a panel of clinically imatinib-resistant BCR-ABL mutants, including the P-loop mutants E255K and Q252H, activation loop mutant H396P, catalytic segment mutant M351T, and mutant F317I in the ATP binding region  over DDR1 . This observation points out the merit of these ureidobenzothiazoles over the known BCR-ABL inhibitors in achieving exceptional selectivity towards BCR-ABL.On the other hand, to gain certain insights about the kinase selectivity of this array of ureidobenzothiazoles, compounds nst DDR1 . Discoid2a–c displayed good anticancer activity against K-562 cell with GI50 values of 3.39–4.52 µM. On the other hand, the m-trifluoromethylphenyl substituted members 2d–f exerted modest cellular potency (GI50 > 10 µM). These cellular outcomes define the impact of BCR-ABL inhibition on the proliferation of K-562 cells. Moreover, compounds 2a–c showed weak cytotoxic effects over L132 normal cell, as evidenced by high GI50 values (17.86–26.78 µM). In terms of selectivity index (SI), the top potent compounds 2a–c had reasonable SI values spanning in the range of 5.3–6.8, which indicate their selective antiproliferative activity towards cancer cell K-562.Motivated by the promising results of cell-free assay, we examined the antiproliferative activity of target compounds against the BCR-ABL overexpressing human leukaemia K-562 cells, along with L132 normal cell by MTT  assay . Consist2b (AK-HW-90), 2e, and 2f were chosen for broad screening against a library of 60 human cancer cell lines, representing nine types of blood and solid cancers. Employing the highly sensitive sulforhodamine B (SRB) assay2b, 2e, and 2f were tested initially at a single dose of 10 μM, and their detailed activities on the percentages growth of the NCI-60 cell lines were illustrated in AKE-5l.To gain further insights about the anticancer profile of this new ureidobenzothiazole chemotypes, the structures of all final compounds were submitted to the National Cancer Institute . Based on NCI selection criteria, compounds 2b, 2e, and 2f showed superior anticancer potency  rather the lead ureidobenzothiazole AKE-5l. While AKE-5l showed narrow spectrum activity towards only K-562 cell line (% growth = 10.22), the o-methoxyphenyl benzothiazoles 2b, 2e, and 2f exerted broad spectrum anticancer effect against multiple cancer cells  over 55 cancer cells. Since both lead AKE-5l and 2b (AK-HW-90) share N-alkylpiperazine and ureidobenzothiazoles as common structural features, it was evident that replacing oxypicolinamide moiety and propyl spacer of AKE-5l with o-methoxyphenyl and phenyl ring in 2b, respectively, led to dramatic change in the anticancer property from narrow into expansive spectrum covering diverse cancer cells. Next in the activity rank is the 3,5-bis(trifluoromethyl)phenyl derivative 2e, which showed cytotoxic rather cytostatic activity against 21 cancer cells. In contrast to 2b and 2e, compound 2f evinced reasonable cytostatic growth inhibitory activity (% growth less than 40) towards 14 cancer cells. Despite of the modest BCR-ABL inhibitory potency of 2e and 2f, they showed good anticancer activity over NCI-60 cell lines, particularly 2e, which points out the existence of other underlying mechanism(s), beside kinase inhibition, that contribute to the anticancer property of this novel chemotype.Interestingly, all three compounds er cells . Among t2b and 2e fulfilled the NCI criteria, therefore, they were advanced to five-dose testing mode to determine their GI50 (the molar concentration causing 50% growth inhibition), TGI (the molar concentration producing 100% GI) and LC50 . The GI50 values, a measure of compound’s potency, of 2b and 2e along with imatinib2b and 2e showed high potency with one-digit micromolar GI50 values against all examined cell lines. Interestingly, 2b and 2e elicited superior anticancer potencies than imatinib over 55 and 54 cell lines, respectively. Regarding leukaemia, both 2b and 2e outperformed imatinib over five leukaemia cell lines; CCRF-CEM and MOLT-4 cells (ALL), HL-60 (TB) (AML), SR (ALK-positive anaplastic large cell lymphoma), and PRMI-8226 (multiple myeloma). For example, 2b and 2e showed equipotent activity against SR cell with GI50 value of ∼ 1.70 µM. Moreover, the growth of imatinib-insensitive lymphoma CCRF-CEM cell was potently suppressed by 2b (GI50 = 1.81 µM) and 2e (GI50 = 2.43 µM). The ethylpiperazine derivative 2b exhibited significant antileukemic activity over the BCR-ABL positive cell K-562 with GI50 value of 0.293 µM.The promising anticancer activities of compounds 2b and 2e possess superior potency to imatinib over seven of the tested NSCL cancer cells. For instance, compounds 2b and 2e displayed GI50 values of 1.47 µM and 1.19 µM against HOP-92 cell line, respectively, with 9–11 folds better potency than imatinib (GI50 = 13.34 µM). The ethylpiperazine member 2b slightly surpassed the potency of its 3,5-bis-trifluoromethyl congener 2e over six cell lines of NSCL cancer panel. Compound 2b exerted equipotent anticancer effects (GI50 ∼ 1.76 µM) over NCI-H460 and NCI-H522 cell lines.From another perspective, it was documented that imatinib could ameliorate the hypoxic circumstances in non-small cell lung cancer (NSCLC)2b and 2e elicited considerable higher anticancer activities than imatinib over all tested colon cancer cell lines. The growth of HT29, the most imatinib-responsive colorectal cancer cell, was strongly inhibited by 2b and 2e, with GI50 values of 1.31 µM and 2.15 µM, respectively. While imatinib exerted modest anticancer activity over COLO205, HCC-2998, HCT-116 and multidrug-resistant HCT-15 colon cancer cells (GI50 = 12.59–21.23 µM), 2b and 2e showed single-digit micromolar GI50 values spanning in the range of 1.07–2.65 µM. Such promising cellular findings recommend further investigations of 2b and 2e as potential candidates for colorectal cancer.On the other hand, imatinib has shown beneficial clinical effects for the treatment of certain metastatic/inoperable gastrointestinal stromal tumours (GIST)2b and 2e were remarkably superior to imatinib across all six examined brain cancer cell lines. Moreover, 2b and 2e exerted good anticancer effects against the temozolomide (TMZ)-resistant cell lines SF-295, SNB-75, SNB-19, and SF-268 gliomas. For instance, the ethyl piperazine member 2b suppressed the growth of the SNB-19 cell line with a GI50 value of 2.03 µM. Referring to melanoma, both 2b and 2e showed comparable potencies against all tested melanoma cell lines, surpassing imatinib, with GI50 values of 1.54–2.51 µM.Since glioblastoma (GBM), the most aggressive form of brain tumours2b and 2e have significant anticancer activity outperforming imatinib over all tested ovarian cancer cells, except OVCAR-5. Particularly, 2b showed GI50 values less than 2.0 µM over all cell lines. Of special interest, ureidobenzothiazoles 2b and 2e exerted appreciable potency towards the paclitaxel-resistant NCI/ADR-RES cell line, with GI50 values of 1.72 µM and 2.37 µM, respectively.Moreover, imatinib efficacy in the treatment of ovarian cancers as a combination with docetaxel was reported2b and 2e, they elicited broad-spectrum activities over multiple cell lines originating from different cancer tissues. As an example, multidrug-resistant (MDR) UO-31 and RXF-393 renal cells showed high sensitivity to 2b (GI50 = 1.52 µM and 1.70 µM) and 2e (GI50 = 1.79 µM and 2.19 µM), respectively. Moreover, the top active member 2b exhibited pronounced anticancer potencies over the prostate cancer cell PC-3, MCF7, and triple-negative breast cancer cell MDA-MB-468 with GI50 values of 1.79, 1.31, and 2.17 µM, respectively.Beside the aforesaid-targeted cellular potencies of 50 values) of ureidobenzothiazoles 2b and 2e  into the catalytic kinase domain of wild-type BCR-ABL (PDB code: 2GQG)2b showed tight binding within the ATP binding site of both BCR-ABLWT and BCR-ABLT315I kinases, as demonstrated by the formation of crucial three hydrogen bonds (HB) with hinge region residue Met318. The ring nitrogen of the benzothiazole scaffold was engaged in HB with amide hydrogen of Met318, while the two NH hydrogens of urea moiety formed two HBs with the amino carbonyl oxygen of Met318. In addition, attractive charge interaction was noticed between the terminal nitrogen of ethyl piperazine and the side chain carboxyl group of Glu329 at the solvent-accessible channel of the BCR-ABL kinase. Moreover, the methoxy group shared carbon-hydrogen bonds with the side chain hydroxyl group of Thr315 in BCR-ABLWT. However, in the case of BCR-ABLT315I, due to steric hindrance with Ile315, which has a larger residual volume than Thr315, such carbon-hydrogen interactions were lacking. Instead, π-alkyl interactions were observed between the methoxy group and the side chain phenyl group of the Tyr253 residue . Besides, in the case of BCR-ABLWT, π-sulphur interaction was noticed between the sulphur atom of benzothiazole and the phenyl group of the Tyr253 residue. The aforementioned binding interactions might justify the sub-nanomolar potency of 2b against both BCR-ABLWT and its T315 mutant.To get insights about the binding mode of this new set of ureidobenzothiazoles, molecular docking for the most potent candidate 2b was predicted by SwissADME online platform2b was found to be in the pink region  with favourable drug-like properties .The bioavailability of compound 2a–f in an effort to improve the BCR-ABL inhibitory activity of our previously reported lead AKE-5l. Two major structural modifications of AKE-5l were undertaken herein, replacing the 6-oxypicolinamide moiety and the propyl spacer of AKE-5l with o-methoxyphenyl and phenyl ring, respectively. Among the target compounds, both 2a and its ethylpiperazine congener AK-HW-90 (2b) stood as the most potent members with IC50 values of 2.0 and 0.65 nM against BCR-ABLT315I, respectively, surpassing the activity of lead AKE-5l (IC50 = 39.9 nM). Furthermore, AK-HW-90 (2b) potently inhibited the activity of a panel of imatinib-resistant mutants with single digit nanomolar IC50 values. On the cellular level, in contrast to the narrow spectrum anticancer activity of the lead AKE-5l, compound AK-HW-90 (2b) exerted considerable broad-spectrum anticancer activity across multiple cell lines including the CML K-562 cell. In addition, AK-HW-90 (2b) surpassed the activity of imatinib over more than 50 cancer cell lines with single digit micromolar GI50 values. Molecular docking study of AK-HW-90 (2b) was conducted to get insights about its postulated binding mode with BCR-ABLWT and BCR-ABLT315I. Considering the cell-free and cell-based assay results, compound AK-HW-90 (2b) may serve as a promising chemotherapeutic candidate for CML and other cancer types.In the current investigation, we report the design and synthesis of new phenyl-ureidobenzothiazoles Click here for additional data file.