Nepeta crispa essential oil against vertebrate L929 and invertebrate Sf9 cell lines were investigated.Pesticides are used as essential tools to control vector-borne diseases and agricultural pests and maintain quality and quantity crop production. Scientists attempt to use derived plant natural products due to environmental safety and low mammalian toxicity. Therefore, the cytotoxicity of malathion and 3 cells were placed into the wells of a 96-well plate experiments. Then appropriate concentrations of malathion and N. crispa essential oil added to the wells. The cells were allowed to grow for 3-5 days and estimated the cell numbers. Control cell wells contained only cells with DMSO. All treatments and controls repeated at least four replicates.About 2×103 cells were placed into the wells of a 96-well plate experiments. Then appropriate concentrations of malathion and N. crispa essential oil added to the wells. The cells were allowed to grow for 3-5 days and estimated the cell numbers. Control cell wells contained only cells with DMSO. All treatments and controls repeated at least four replicates.About 2×10Nepeta crispa essential oil had negative effects on the Sf9 cell viability with the differences that derived plant natural products containing environmentally friendly and readily biodegradable derivatives, hydrolyzing rapidly in nature and nearly having no destructive effects on mammals and environment.Plant essential oil not only had no negative effects but also had boosting effects on the L929 cell viability.  At present, pesticides with increasingly global marketing are used as an essential tool to control of vector-borne diseases and agricultural pests and maintain quality and quantity crop production. They also play a key role in the prevention and control of infectious diseases such as malaria, dengue, and filariasis . DespiteNepeta is a genus of Lamiaceae family that has been spread in many parts of the world, including Asia, Europe and North Africa. About of 280 annual or perennial worldwide species of Nepeta genus, there are 79 species present in Iran with of 38 species of native to the country [Nepeta species are widely used as antispasmodic, anti-asthma, sedative, treatment of various digestive, neurological and respiratory diseases [Nepeta genus species [Nepeta crispa Willd.  is one of the most aromatic plants in Iran which is popular in Iranian traditional medicine, especially people of Hamadan province. Nepeta crispa is autochthonous of Hamadan climate having insecticidal activity, and antimicrobial and antifungal properties [N. crispa, but a simultaneous comparative study about the cytotoxicity of N. crispa against the cell lines of invertebrates and vertebrates would be of particular importance. Therefore in this study, we compared the cytotoxicity of N. crispa essential oil with malathion against vertebrate and invertebrate cell lines.Many Iranian wild flowers have medicinal and insecticidal properties. Several studies have been conducted on Lamiaceae family due to their toxic effects on various insect species. The family of Lamiaceae have high diversity and distribution in flora of Iran containing 46 genera and 420 species and sub-species . Nepeta  country , 23. In diseases -26. It'sdiseases -29. Many species -32. Nepeoperties , 34. ManProviding of insecticide and plant materials: liquid technical grade of malathion (95%) were purchased from India's Haramba Company. The aerial parts (foliage) of Nepeta crispa during their flowering stage were collected from Avicenna Medicinal Herbs Research Center, Hamadan Province of Iran in June 2017. The plant was confirmed by a voucher specimen (no. 72) in the Department of Pharmacognosy, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran.Plant essential oil isolation: a total of 1000 g powder of shade-dried aerial parts of N. crispa were subjected to hydrodistillation using a clevenger-type apparatus for 4h. The essential oil was dehydrated over anhydrous sodium sulphate and transferred into amber-colored vials to store in a refrigerator at 4ºC for further work.Invertebrate cell line: Sf9 cell line which was derived from the ovary of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) was provided from National Cell Bank of Pasture Institute of Iran. Sf9 cell line is routinely cultured and maintained at 27ºC in 5ml of Grace's insect cell culture medium in 25cm2 culture flasks, enriched with 10% fetal bovine serum at Pasture Institute of Iran. The cell doubling time for this cell was found to be 18-24h under optimum conditions. Cells were sub-cultured every 3 days [y 3 days .Vertebrate cell line: L929 vertebrate cell line which was derived from mouse fibroblast cells used for this study. It was provided from National Cell Bank of Pasture Institute of Iran. It was maintained at 37ºC in 3ml of DMEM Media (Gibco®) in 25cm2 culture flasks, enriched with 10% fetal bovine serum and buffered with 4% sodium bicarbonate in an atmosphere of 5% carbon dioxide. The doubling time for cultures was approximately 24h and the cell was sub-cultured every 6 day [ry 6 day .Cell bioassay: technical grade of malathion (95%) was dissolved with ethanol 96% to prepare the concentrations of 10-10, 10-9, 10-8, 10-7, 10-6, 10-5, 10-4 and 10-3 μg-μL containing 0.000095, 0.00095, 0.0095, 0.095, 0.95, 9.5, 95 and 950 μg-μL, respectively. Herbal essential oil (0.1 mg) of Nepeta crispa was dissolved with 1ml of DMSO (dimethyl sulfoxide) due to hydrophobic properties and then diluted with sterilized distilled water to prepare the concentrations of 10-10, 10-9, 10-8, 10-7, 10-6, 10-5, 10-4 and 10-3 ng-μL containing 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng-μL, respectively. To consider the cytotoxicity of malathion and N. crispa essential oil against L929 and Sf9 cell lines, about 2×103 cells per 100 μl of culture medium were placed into the wells of a 96-well plate experiments of treatments and then appropriate concentrations of N. crispa essential oil and malathion added to the wells. We allowed the cells to grow for 3-5 days, and estimated the number of cells as described. Control cell wells contained only cells with 1 μl-mL of DMSO. All treatment and control experiments repeated at least four replicates.Estimation of cell number: the base method for cell estimation is the Mossman method, which uses 3--2,5-difenyltetrazolium bromide . MTT is a quantitative coloring for living cells and cell proliferation, and it's a known method for invitro cytotoxicity which measures the active metabolism of the cells. In this coloring solution, dehydrogenase enzyme reduced the MTT and produced blue formazan. The wells of 96-well plates containing L929 and Sf9 cell lines were incubated with 10 μ MTT for 3h at 36ºC and 27ºC, respectively. After the blue formazan and cells settled out and the supernatant was removed, 100 μ of DMSO was added to any well of 96-well plate, shaked for 15 minutes and then the absorbance of the solution read at 492 nm using ELISA reader [Statistical analysis: IBM SPSS statistics data editor version 24 was used for any statistical analyses. Wilcoxon signed ranks-test was used for comparing cytotoxicity of malathion and essential oil of Nepeta crispa between control and treatments, and treatments against L929 and Sf9 cell lines. P < 0.05 was considered significant. The trends of malathion and N. crispa essential oil cytotoxicity against L929 and Sf9 cell lines was estimated by Microsoft Office Excel 2013. The trends were drawn by clicking on graph line distribution and selecting “add trendline” option using Nasirian and Salehzadeh  style [b) style , 37-40.Statistical analysis: IBM SPSS statistics data editor version 24 was used for any statistical analyses. Wilcoxon signed ranks-test was used for comparing cytotoxicity of malathion and essential oil of Nepeta crispa between control and treatments, and treatments against L929 and Sf9 cell lines. P < 0.05 was considered significant. The trends of malathion and N. crispa essential oil cytotoxicity against L929 and Sf9 cell lines was estimated by Microsoft Office Excel 2013. The trends were drawn by clicking on graph line distribution and selecting “add trendline” option using Nasirian and Salehzadeh  style [b) style , 37-40.Malathion cytotoxicity:Nepeta crispa (-μL) and essential oil of Nepeta crispa (ng-μL) against L929 and Sf9 cell lines. The cytotoxicity of malathion against L929 and Sf9 cell lines were gradually increased with a relatively low decreasing slope in accordance with malathion concentrations from 10-10 to 10-3 μg-μL        . Althoug= 0.068) . While t control . Wilcoxo > 0.05) , even thll lines .N. crispa essential oil:Comparison between cytotoxicity of malathion and  the cytotoxicity of malathion against L929 and Sf9 cell lines were gradually increased with a relatively low decreasing slope in accordance with malathion concentrations. The cytotoxicity of N. crispa essential oil against L929 cell lines were gradually decreased with a moderately increasing slope in accordance with N. crispa essential oil concentrations. While the cytotoxicity of N. crispa essential oil against Sf9 cell lines were strongly increased with an intensive increasing slope in accordance with N. crispa essential oil concentrations  . While there was a significant difference at P < 0.001 level between treatments of 10-10 to 10-7 malathion concentrations with N. crispa essential oil against Sf9 cell lines (trations . Wilcoxo < 0.05) . Even thll lines .-5 to 10-3 and 10-8 to 10-3 malathion concentrations with control group against the L929 and Sf9 cell lines, respectively at P < 0.05 or P < 0.001 levels  was 1,000 folds lower than malathion concentrations (μg-μL). The viability of Sf9 cell lines which were exposed to concentrations of 10-9 and 10-7 to 10-3N. crispa essential oil was lower than control group and decreased with increasing N. crispa essential oil concentrations  is one of the most aromatic plants in Iran. N. crispa is autochthonous of Hamadan climate. A simultaneous comparative study about the cytotoxicity of N. crispa against the cell lines of invertebrates and vertebrates would be a particular of importance. Therefore, the cytotoxicity of N. crispa essential oil and malathion against L929 cell line of vertebrates and Sf9 cell line of invertebrates were investigated. Based on the results of the study, malathion had negative effects on the viability of both L929 and Sf9 cell lines. Unlike malathion, essential oil of N. crispa not only did not have negative effects on the viability of L929 cell lines, but also have boosting effects on the viability of L929 cell lines. Significant differences are also observed between treatments of 10-10 to 10-3N. crispa essential oil concentrations with treatments of malathion 10-10 to 10-3 concentrations and control group against the L929 cell lines by Wilcoxon signed-ranks test confirming this fact. Like malathion, essential oil of N. crispa had negative effects on the viability of Sf9 cell lines with the differences that the natural products deriving from plants containing environmentally friendly and readily biodegradable derivatives, hydrolyzing rapidly in nature and nearly having no destructive effects on mammals, humans or the environment. In addition, the application of N. crispa essential oil concentrations was extremely lower than malathion concentrations. On the other hand there is no concern about plant essential oil potential side effects like direct toxicity to users, environmental pollution, ozone depletion, pesticide residues, and toxicity to non-target organisms with application of the natural products deriving from plants. We will not also encounter probably some type of cancers or muscle fatigue, neurological diseases, and psychotic disorders by applying the derived natural products of plants. As well as maybe no longer encounter to a substantial problem of the vector and pest management programs due to insecticide resistance in arthropods of the vectors of diseases and agricultural pests.Pesticides are used as an essential tool to vector-borne diseases and agricultural pests, and maintain quality and quantity crop production. With great concern about environmental problems and human health of synthetic pesticides, scientists have attempted to use natural products derived from plants. Scientists attempt to use derived plant natural products due to environmental safety and low mammalian toxicity;Nepeta crispa  is one of the most aromatic plants in Iran which is popular in Iranian traditional medicine.Plant essential oil not only had no negative effects but also had boosting effects on the L929 cell viability.Nepeta crispa essential oil had negative effects on the Sf9 cell viability with the differences that derived plant natural products containing environmentally friendly and readily biodegradable derivatives, hydrolyzing rapidly in nature and nearly having no destructive effects on mammals and environment.The authors declare no competing interests.


NFKB1, BAG3, MCL1, GADD45A, and HRK in CD133+ CSCs, which, if targeted, might increase the sensitivity of NSCLC to MSC-TRAIL-mediated inhibition. As such, our findings add credibility to the utilization of MSC-TRAIL for the treatment of NSCLC through targeting of CD133+ CSCs.Mesenchymal stem cells (MSCs) are emerging as vehicles for anti-tumor cytotherapy; however, investigation on its efficacy to target a specific cancer stem cell (CSC) population in non-small cell lung cancer (NSCLC) is lacking. Using assays to evaluate cell proliferation, apoptosis, and gene expression, we investigated the efficacy of MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) to target and destroy CD133+ (prominin-1 positive) NSCLC-derived CSCs. Characterization of TRAIL death receptor 5 (DR5) revealed that it was highly expressed in the CD133+ CSCs of both H460 and H2170 cell lines. The human MSC-TRAIL generated in the study maintained its multipotent characteristics, and caused significant tumor cell inhibition in NSCLC-derived CSCs in a co-culture. The MSC-TRAIL induced an increase in annexin V expression, an indicator of apoptosis in H460 and H2170 derived CD133+ CSCs. Through investigation of mitochondria membrane potential, we found that MSC-TRAIL was capable of inducing intrinsic apoptosis to the CSCs. Using pathway-specific gene expression profiling, we uncovered candidate genes such as  Lung cancer is the most frequently diagnosed cancer worldwide, accounting for more than 1.7 million 18.4%) of total cancer deaths in 2018 . The ant% of totaCASP8/10 and intrinsic apoptosis through cytochrome C release from the mitochondria. However, due to its short half-life and likelihood to be eliminated through renal filtration, TRAIL needs a delivery system to be effective  assay (proliferation assay) performed in the NSCLC cell lines  in the presence of different rhTRAIL concentrations showed that each of these cell lines responded differently to the rhTRAIL treatment . The expression of CD133+ in all of the NSCLC cell lines was low (~0.1% expression), consistent with a previous report that identified CSCs in lung cancer samples A 80]. T. T80]. Tp < 0.001) were noted in H460- and H2170-derived CD133+ CSCs, reaching an average diameter of 130.47 ± 22.2 and 156.66 ± 36.48 μM, respectively, compared to the CD133− (45.71 ± 17.11 and 66.45 ± 15.83 μM) and unsorted (56.99 ± 19.28 and 62.88 ± 13.84 μM) cell lines (p < 0.001) was detected in the CD133+ population derived from H460 and H2170 with an average sphere number of 138 ± 9.5 and 199 ± 11.3, compared to a lower number of spheres in the CD133− (45.0 ± 1.4 and 113 ± 2.8) population . Significantly bigger spheres (ll lines A. In addpulation A. Howevep < 0.001) number of colonies with 150.0 ± 12.9, 165.8 ± 10.4, and 96.6 ± 3.8 than CD133− with only 58.14 ± 4.9, 71.2 ± 9.0, and 53.6 ± 8.2 colonies detected, respectively (CD133+ NSCLC-derived CSCs and controls (CD133− and unsorted) were seeded at a very low density (1000 cells in 2 mL) in a six-well plate. After 14 days, formed colonies were stained and manually counted. The H460-, H2170-, and A549-derived CD133+ CSC population presented a significantly higher (ectively B.Reports showed that high ALDH activity is an important indicator for chemoresistance . As CSCsp < 0.001) of CD133+ CSC viability from both H460 and H2170 was detected when these cells were co-cultured with MSC-TRAIL starting from a ratio of 1:1, and the cytotoxic effect of MSC-TRAIL was more evident as the ratio increased (The sorted (CD133+ and CD133−) and unsorted NSCLC cell lines were co-cultured with MSCs (MSC-TRAIL or MSC-EV) at different NSCLC-cell-to-MSC ratios  in a 96-well tissue culture plate for 72 h, and rhTRAIL was used as a positive control for the treatment. Under phase-contrast microscopy, the formation of apoptotic bodies and dead cells was clearly seen in both H460- and H2170-derived CD133+ cells cultured with MSC-TRAIL when compared to the MSC-EV (cultured at a 1:1 NSCLC-cell-to-MSC ratio), depicted in ncreased B. Howevencreased B.p < 0.001) was also noted in CD133− and unsorted cells derived from H460 and H2170 co-cultured with MSC-TRAIL than in the control (MSC-EV) culture, suggesting that both NSCLC cell lines were highly sensitive to TRAIL-mediated killing  in CD133+ CSCs, both MSC-EV and MSC-TRAIL were co-cultured with the sorted (CD133+ and CD133−) and unsorted NSCLC cell lines  at an NSCLC-cell-to-MSC ratio of 1:1 for 72 h . An incr killing B. Furthep < 0.001) percentage of Sytox-Green-positive dead cells in the CD133+ population of H460, H2170, and A549 cultured with MSC-TRAIL  than that with MSC-EV  was seen  and CD133+ NSCLC -derived CSCs, as well as the CD133− and unsorted cells, were co-cultured  for 72 h and assayed for mitochondrial membrane potential (ΔΨ) . The per2 profiler PCR arrays  to identify apoptotic genes that are uniquely regulated in CD133+ H460-derived CSCs by MSC-TRAIL. The CSCs were subjected to different treatments  for 48 h, and untreated CSCs were used as a baseline control . The gene expression depicted in the heat map between the treatments (MSC-TRAIL and rhTRAIL) and controls (MSC-EV and untreated) was consistent, indicating that the regulation of apoptotic genes by TRAIL was specific (CASP1), baculoviral-encoded inhibitor of apoptosis/IAP repeat containing 3 (BIRC3), tumor necrosis factor receptor superfamily member 25 (TNFRSF25), cluster of differentiation 40 (CD40), TNFRSF21, B-cell lymphoma 2/BCL2-associated athanogene 3 (BAG3), induced myeloid leukemia cell differentiation protein (MCL1), nuclear factor kappa beta subunit 1 (NFKB1), fas cell surface death receptor (FAS), and cell-death-inducing DNA fragmentation factor-α/DFFA-like effector B (CIDEB) were significantly upregulated , caspase 5 (CASP5), harakiri BCL2-interacting protein (HRK), and TNFRSF10B) were highly downregulated, as indicated in p ≤ 0.05, fold-change ≤ 2.0), and by the blue dots in CASP3, BCL2-associated X apoptosis regulator (BAX), BCL2-like 1 (BCL2L1), BCL2-interacting protein 2 (BNIP2), and Nod-like receptor/NLR family apoptosis inhibitory protein (NAIP)) were downregulated with a fold-change ≤ 1.0  formation /luminescence value of H2170 (untreated).Since both A549 and H460 constitutively produce luciferase, the viability of these cells after treatment was reflected by the activity of luciferase to convert luciferin into oxyluciferin, which can be detected as luminescence. Briefly, sorted (CD133+ and CD133−) and unsorted A549 and H460 cells were initially seeded in a 96-well tissue culture plate at 1.0 × 104 in 200 μL of medium) in a 24-well tissue culture plate. Next, treatments of MSC-EV, MSC-TRAIL (5.0 × 104 in 200 μL of medium), or rhTRAIL  were added to the wells. After 72 h, cells were harvested by trypsinization and collected by centrifugation. Annexin V binding buffer (100 μL) was added with 1 μL of annexin V–FITC antibody and incubated at 4 °C in the dark. For dead cell analysis, 1 μL of Sytox Green  was added to 500 μL of cell suspension and directly used for analysis. Stained cells were subjected to flow cytometric analysis, and 10,000 events were collected using the FACS Calibur instrument (Becton Dickinson BD) and analyzed using Cell Quest Pro software gated on FL-1 (Becton Dickinson BD).Analysis of apoptosis was evaluated using FITC-conjugated annexin V antibody from BD Pharmingen . In brief, sorted and unsorted NSCLC cell lines  were initially seeded  into a 24-well tissue culture plate. After 72 h of incubation, cells were trypsinized and collected by centrifugation, before being suspended in 250 μL of JC-1 stain (diluted from stock) and incubated for 15 min in the dark. Stained cells were then washed twice with assay buffer and pelleted by centrifugation. Collected cells were suspended in DPBS containing 2% FBS and kept on ice. Analysis was performed using the FACS Calibur instrument (Becton Dickinson BD) and gated at FL-1 (green fluorescence) and FL-2 (red fluorescence) channel.To look into the activation of the intrinsic apoptosis in sorted and unsorted NSCLC cell lines post-treatment, we conducted mitochondria membrane potential (ΔΨ) analysis using the BD MitoScreen . Sorted and unsorted NSCLC cell lines were seeded with MSC-TRAIL, MSC-EV at 1:1 ratio, or rhTRAIL . The human apoptosis RT² profiler PCR array profiles the transcriptional level using quantitative RT-PCR of 84 critical genes categorically divided into anti-apoptosis, regulators of apoptosis, death domain receptors, and caspases. Sorted and unsorted NSCLC cell lines were seeded with MSC-TRAIL, MSC-EV (1:1 ratio), or rhTRAIL  in a 24-well tissue culture plate. After 48 h, total RNA was extracted from samples and on-column DNA digestion was performed during the extraction process. RNA (0.5 μg) was added into the genomic DNA elimination mix to a total of 10 μL. The sample was incubated at 42 °C for 5 min and then on ice for 1 min. Reverse transcription mix (10 μL) was then added, and the sample was incubated at 42 °C for 15 min and 95 °C for 5 min. After the reaction time, 91 μL of RNAse-free water was added to make a total of 111 μL of cDNA synthesizing reaction. The PCR component mix was prepared by adding 102 μL of cDNA synthesizing reaction to 2× RT2 SYBR Green Mastermix and RNase-free water to a total volume of 2700 μL. Subsequently, 25 μL of the reaction was pipetted into each well of the 96-well PCR array plate and centrifuged at room temperature for 1 min at 1000× g . The PCR cycling process  was performed using LightCycler 480 . Analyses of data were performed using web-based analysis software [The 84 key genes and biological pathways involved in cell death activation by TRAIL were analyzed in CD133+ H460-derived CSCs using the Apoptosis RTsoftware .t-test with p < 0.05 considered statistically significant. Comparison between groups was performed using one-factor analysis of variance (ANOVA). Analyses were performed using Excel 2010, version 14.0 .Data are presented as means ± standard deviation (SD) of three independent experiments. Comparison between two groups was performed using the two-tailed NFKB1, BAG3, MCL1, GADD45A, and HRK as potential anti-cancer genes that could increase sensitivity of NSCLC to MSC-TRAIL therapy. To further understand the mechanism of TRAIL resistance in the CD133+ CSCs, functional assays incorporating MSC-TRAIL and a knockdown or sensitized model of the CSCs and NSCLC cell lines can be performed in the future.Collectively, our results revealed that NSCLC-derived CD133+ CSCs can be effectively targeted using MSC-TRAIL through the extrinsic and intrinsic apoptosis pathways. While common chemotherapy may not be effective in destroying CSCs and tumor cells in general, MSC-TRAIL could serve as a better alternative or as a complement to the treatment in NSCLC patients. Finally, through gene expression analysis, we identified signaling molecules that might contribute toward TRAIL and MSC-TRAIL resistance in CD133+ CSCs, and uncovered


Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) in four human cancer cell lines, A549, SCC-15, SH-SY5Y, and CACO-2, and investigate its impact on the production of reactive oxygen species (ROS) and the apoptotic process as well as cytotoxicity and metabolism in these cell lines. The cell lines were exposed to increasing concentrations (1 nM to 100 µM) of the studied compound for 6, 24, and 48 h, and later, ROS production, cell viability, caspase-3 activity, and cell metabolism were examined. The obtained results showed that the studied compound decreased the production of ROS, increased the release of lactate dehydrogenase, and decreased cell metabolism/proliferation in all the five cell lines at micromolar concentrations. Interestingly, over a wide range of concentrations (from 1 nM to 100 µM), Les-236 was able to increase the activity of caspase-3 in BJ (after 6 h of exposure), A549, CACO-2, and SCC-15 (after 48 h of exposure) cell lines which could be an effect of the activation of PPARγ-dependent pathways.4-thiazolidinones, which are privileged structures in medicinal chemistry, comprise the well-known class of heterocycles and are a source of new drug-like compounds. Undoubtedly, the 5-bulky-substituted-2,4-thiazolidinediones - a class of antihyperglycemic glitazones, which are peroxisome proliferator-activated receptor gamma (PPARγ) agonists, are the most described group among them. As there are various chemically distinct 4-thiazolidinones, different subtypes have been selected for studies; however, their main pharmacological profiles are similar. The aim of this study was to evaluate the anticancer activity of 5 Undoubtedly, the 5-bulky-substituted-2,4-thiazolidinediones, a class of antihyperglycemic glitazones, which are peroxisome proliferator-activated receptor gamma (PPARγ) agonists, are the most described group among them3. As there are various chemically distinct 4-thiazolidinones, different subtypes have been selected for investigations; however, their main pharmacological profiles are similar and they are known to exhibit anticancer, anti-inflammatory, antiviral, and antimicrobial activity. Moreover, different 4-thiazolidinones -4-thiazolidinone, etc.), and their related (hydantoin) cores are considered as bioisosteres. 5-ene-2-amino(imino)-4-thiazolidinones represent one of the most studied subtypes of 4-thiazolidinones. The importance of C5-ene fragment had been proved in many studies6. Moreover, conjugation of the C5 exocyclic double bond to the C4 carbonyl group makes these compounds electrophilic and potentially reactive in Michael addition. This is one of the reasons why 5-ene-4-thiazolidinones, especially rhodanines, are called as pan-assay interference compounds9. This concept is still under debate; however, many useful properties of Michael acceptors have been proposed10: Michael acceptors, among the most effective activators of Nrf2, open new perspectives in the treatment of inflammation and cancer11. They act as inducers of phase 2 enzymes and a group of inducible proteins12 and covalent inhibitors13. Moreover, Michael acceptors function as reactive electrophilic species, one of the key signaling species14. Among the 5-ene-2-amino(imino)-4-thiazolidinones, active anticancer agents which are capable of inhibiting the growth of cancer cells were identified both in screening campaigns and small-scale studies21. Many ligands exhibiting high affinity to the known anticancer targets were also described, including integrin αVβ3 antagonists22, inhibitors of CDK1/cyclin B24, PPAR antagonists25, estrogen-related receptor-α modulators26, SHP-2 inhibitors27, PTP1B inhibitors28, DNA-binding agents 29, and so on. Very few attempts have been made to outline the main molecular modes of action of 5-ene-2-amino(imino)-4-thiazolidinones30. Analyses of the molecular mechanisms of their action revealed their role in the following processes: induction of apoptosis, cell cycle arrest33, PPAR-related pathways25, inhibition of necroptosis35, and modulation of Red/Ox signaling and production of reactive oxygen species (ROS). Sometimes contrasting results are presented for such compounds; for example, one study reported that these compounds decrease the level of ROS36, whereas some other studies reported that the action of these compounds is associated with increase in the production of ROS37. The PPAR-related mechanism behind the anticancer effect was actively discussed following the identification of the role of ligand-activated transcription factor. PPARγ regulates diverse functions, such as the development of fat cells and their capacity to store lipids, expression of genes related to metabolic processes, inflammation, cell migration, apoptosis, and cell sensitivity to insulin. Moreover, in 1997, the first published reports highlighted PPARγ as a novel cancer therapeutic target regulating the differentiation of cancer cells39. Glitazones induce a variety of favorable changes in several malignancies, including liposarcoma and cancers of various organs such as breast, colon, pancreas, and prostate41. In vitro exposure of tumor cells to high doses of troglitazone and ciglitazone was found to lead to cell cycle arrest, apoptosis, and redifferentiation44, suggesting the involvement of PPARγ signaling pathways. Furthermore, the in vivo anticancer efficacy of troglitazone was demonstrated in a few clinical studies that involved patients with liposarcomas or prostate cancer45. The precise mechanism of antiproliferative activities of PPARγ agonists remains unclear43; increasing evidence indicates that 4-thiazolidinones mediate PPARγ-independent antitumor effects by targeting a wide range of signaling pathways controlling the proliferation and survival of cancer cells46. Analyses of the effects of 5-ene-2-amino(imino)-4-thiazolidinones also revealed the importance of these metabolic pathways in anticancer activity2. Additionally, it should be highlighted that anticancer agents such as 5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5 H)-thiazolone (MMPT) and 5--2-(phenylimino)-1,3-thiazolidine are also effective in the treatment of multidrug-resistant cancer47. The structure of MMPT has been considered as a “pharmacophore model” for further optimization21, and a set of structural requirements were proposed: an unsubstituted N3 atom in the main core, a C5 p-substituted-arylidene moiety, and a C2-arylamino fragment which can be substituted in different positions18. Moreover, MMPT analogs were found to exert anti-inflammatory activity through COX/LOX inhibition48. Other different activities of the related compounds are also being studied. The pharmacological profiles of compounds with anticancer effect and compounds exerting a combination of anticancer, antioxidation, anti-inflammatory49, and antiprotozoal50 effects are gaining interest. The present work is an extension of our ongoing efforts in the search for new 4-thiazolidinone-based anticancer agents51. Therefore, the aim of this study was to evaluate the anticancer activity of 5Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) in four human cancer cell lines A549, SCC-15, SH-SY5Y, and CACO-2, and investigate its impact on ROS production and the apoptotic process as well as cytotoxicity and metabolism in these cell lines  by 17.10–42.83% compared to control Fig. . Similar2O2), ROS production stimulated by H2O2 was significantly decreased by Les-236. The strongest effect of Les-236 was observed in BJ cells and the weakest in A549 cells  were calculated for LDH release to determine the dose-dependent effect on cell lethality . In the SCC-15 cell line, Les-236 increased caspase-3 activity at 10, 50, and 100 µM concentrations . The strongest increase in caspase-3 activity by Les-236 was observed in the BJ cell line where Les-236 increased caspase-3 activity at all studied concentrations (1 nM to 100 µM) by 111.42–495.56% compared to control Fig. . After 2After 6 h of exposure of the BJ cell line to 1 and 10 µM concentrations of Les-236, there was an increase in the expression of PPARγ by 25.76% and 17.31%, respectively. In the A549 cell line, 1 and 10 µM concentrations of Les-236 similarly increased expression of PPARγ by 36.03% and 23.33%, respectively. In the SH-SY5Y cell line, an increase in PPARγ expression was observed after treatment with 1 µM Les-236 (increased by 25.03% compared to control). In the SCC-15 cell line, a decrease in PPARγ expression was observed after treatment with 1 µM Les-236 (decreased by 12.26% compared to control). In the CACO-2 cell line, 1 and 10 µM concentrations of Les-236 decreased the expression of PPARγ by 21.67% and 36.25%, respectively  and the antagonist (GW9662) of PPARγ decreased protein expression of PPARγ . Cell co-treatment with Les-236 and rosiglitazone or Les-236 and GW9662 did not affect expression of PPARγ compared to rosiglitazone alone or GW9662 alone, respectively. Similarly, in CACO-2 cells, 1 µM Les-236 did not affect expression of PPARγ. Rosiglitazone increased PPARγ protein expression by 5.83 ng/mL. GW9662 decreased PPARγ protein expression in CACO-2 cells by 2.03 ng/mL. Les-236 prevented decrease in PPARγ protein expression caused by GW9662. In SCC-15 cells, 1 µM Les-236 decreased in PPARγ expression by 4.47 ng/mL. Interestingly, GW9662 increased PPARγ expression by 6.96 ng/mL. Les-236 decreased expression of PPARγ stimulated by GW9662 (decreased by 12.42 ng/mL compared to GW9662 stimulated cells). After the exposure of A549 cells to 1 µM Les-236, no changes in the PPARγ protein expression were observed. Cell co-treatment with Les-236 and rosiglitazone increased PPARγ protein expression compared to the rosiglitazone treated group (increased by 1.04 ng/mL). After 48 h of exposure of the SH-SY5Y cell line to 1 µM Les-236, a decrease in the PPARγ protein expression was observed (decreased by 0.63 ng/mL). Cell stimulation with rosiglitazone and GW9662 decreased PPARγ expression by 0.31 and 0.70 ng/mL, respectively compared to control. Cell co-stimulation with Les-236 and rosiglitazone or Les-236 and GW9662 enhanced the effect of rosiglitazone or GW9662 alone ; however cell co-treatment with Les-236 and rosiglitazone did not affect KI67 expression, compared to the rosiglitazone treated group. GW9662 did not affect KI67 protein expression in A549 cells but cell co-treatment with Les-236 and GW9662 decreased expression of KI67 . In the SH-SY5Y cell line, 1 µM Les-236 decreased KI67 protein expression by 0.16 ng/mL, compared to control. Cell stimulation with rosiglitazone and GW9662 decreased PPARγ expression by 0.15 and 0.25 ng/mL, respectively compared to control. Cell co-stimulation with Les-236 and rosiglitazone or Les-236 and GW9662 did not affect the expression of KI67 compared to rosiglitazone or GW9662 alone -4-thiazolidinones showed a positive anticancer activity in a previous screening16, we decided to study the anticancer potential of 5Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) in the following four human cancer cell lines: squamous cell carcinoma (SCC-15), lung carcinoma (A549), colon adenocarcinoma (CACO-2), and neuroblastoma (SH-SY5Y). Our experiments showed a decrease in ROS production in all the studied cancer cell lines  and the normal skin fibroblast cell line (BJ) under the action of Les-236. The strongest decrease in ROS production in all the time intervals was observed in CACO-2 cell line, while SH-SY5Y cell lines showed the least sensitivity to Les-236. Moreover, in all studied cell lines, cell co-treatment with H2O2 and Les-236 showed that Les-236 reduced ROS production stimulated by H2O2. Currently, there is evidence that the mechanism of anticancer activity of structurally similar 4-thiazolidinones is associated with increase in the level of ROS37. However, our data suggest a different mechanism for the activity of Les-236. Similar results have been previously obtained by Коbylinska et al. (2016) who studied the activity of 5-heterylidene 2--4-oxo-4,5-dihydro-1,3-thiazoles in rat glioma (C6) cell line53. The compound studied by Коbylinska et al. (2017) also decreased ROS production in human glioma (U251) cell line54. Moreover, decrease in ROS production correlated with decrease in the activity of superoxide dismutase and increase in the activity of catalase53. Due to similiarities in chemical structure between the two compounds, we belived that Les-236 might act in a similar way.The International Agency for Research on Cancer estimated that there were 9.6 million cancer deaths in 2018Our study showed that despite decrease in production of ROS, the Les-236 compound exhibited cytotoxicity. The strongest induction of LDH release was observed in BJ, SCC-15, and SH-SY5Y cell lines. The resazurin reduction assay revealed that Les-236 decreased metabolism/proliferation in all the studied cell lines at micromolar concentrations, with the strongest reduction observed in SH-SY5Y cell line. All the tests showed that neuroblastoma was very sensitive to Les-236, as the compound caused necrosis in the SH-SY5Y cell line.in vitro concentrations (µM) of thiazolidinediones (TZDs), such as ciglitazone, rosiglitazone, pioglitazone, and troglitazone, lead to cell cycle arrest, apoptosis, and redifferentiation55, suggesting a putative link between PPARγ signaling and the antitumor activities of TZDs. The mentioned TZDs were shown to increase LDH release in mouse glioma cells (GL261), C6, human colorectal adenocarcinoma (HT29), and human colon carcinoma (COLO-205) cell lines57, as well as decrease cell metabolism/proliferation in human breast cancer (MCF-7), A549, and human prostate cancer (PC-3) cell lines59. Furthermore, the in vivo anticancer efficacy of troglitazone was demonstrated in a few clinical studies that involved patients with liposarcomas or prostate cancer45. Similar to our study, Коbylinska et al.54 showed that a new derivate of 4-thiazolidinone (Les-3288) decreased cell metabolism/proliferation as revealed by the MTT assay in C6 and U251 cells54. Our previous studies showed that 4-thiazolidinone derivatives  caused LDH release and decrease of resazurin reduction in SCC-15 cell lines37. Moreover, PPARγ silencing reduced the toxicity of related thiazolidinones  which confirms the crucial role of PPARγ receptors in the mechanism of action of 4-thiazolidinone derivatives20.To date, it has been described that high 60. Our study showed that after 6 h of exposure to Les-236, caspase-3 activity increased in the BJ cell line at all studied concentrations. In cancer cell lines , caspase-3 activity was increased only at micromolar concentrations after 6 and 24 h of exposure to Les-236. Interestingly, after 48 h of exposure, Les-236 caused caspase-3 activation at all studied concentrations in A549 (excluding 100 µM which is a highly necrotic concentration), CACO-2, and SCC-15 cell lines. The observation that Les-236 also caused caspase-3 activation in normal skin fibroblasts (BJ) is disturbing. The long duration of exposure needed for apoptosis activation in A549, CACO-2, and SCC-15 cell lines suggests the complex mechanism of action of Les-236 and its promising effect in cancer treatment. Moreover, in A549, CACO-2, and SCC-15 cell lines, apoptosis correlated with increase in expression and/or activation of PPARγ62. Our previous study showed that Les-3377 and Les-3640 increased caspase-3 activity in SCC-15 cells, and silencing of PPARγ prevented the activation of caspase-337.The most important marker of apoptosis is activation of caspase-3 which results in DNA fragmentation, degradation of the cytoskeleton and nuclear proteins, cross-linking of proteins, and finally, formation of apoptotic bodies50) is the most widely used and informative measure of a drug’s efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of the potency of an antagonist drug in pharmacological research. As measured by resazurine reduction, Les-236 has the highest potential to inhibit cell metabolism/proliferation in the SH-SY5Y cell line (0.24 µM) and the weakest in the A549 cell line (2062.00 µM) (Table 50) level is a well-known quantity used in pharmacology and toxicology to calculate the efficacy of a substance from a dose–response curve. It represents the dose at which 50% of the maximum efficacy is observed. EC50 values were calculated for LDH release and caspase-3 activity to determine the dose-dependent effect on lethality to cells. The lowest EC50 for LDH release was for the SH-SY5Y (0.37 µM) cell line. For caspase-3 assay, the lowest EC50 was for the CACO-2 cell line (10.36 µM).The half-maximal inhibitory concentration (ICM) Table . The halin vitro studies, high concentrations (µM) of TZDs, led to cell cycle arrest, apoptosis, and redifferentiation55. However, in some cell lines, such as SCC, A549 and CACO-2, TZDs can stimulate cells proliferation depending on the concentration64. Our experiments show that after a 6 h exposure, Les-236 affects PPARγ mRNA expression in all studied cell lines. Moreover, Les-236 alone or in co-treatment with rosiglitazone or GW9662, affects expression of PPARγ protein in the studied cell lines. In CACO-2, A549, and SH-SY5Y cell lines, Les-236 acts similar to GW9662. In the SCC-15 cell line, Les-236 acts similar to rosiglitazone. Unfortunately, in the BJ cell line, results are not conclusive and we cannot clearly determine whether Les-236 works similar to an agonist or an antagonist of PPARγ. We suggest that in the A549, CACO-2, and SCC-15 cell lines, PPARγ-dependent pathways could be involved in cell death caused by Les-236; however, more studies are needed to confirm this hypothesis.In the second part of our study we showed that after cell exposure to 1 µM Les-236, KI67 protein (considered as a marker of proliferation) expression significantly decreased in BJ and SH-SY5Y cell lines which correlates with decreased resazurine reduction. In the mentioned cell lines, co-stimulation with Les-236 and rosiglitazone (PPARγ agonist) or GW9662 (PPARγ antagonist) did not affect expression of KI67 decreased by Les-236. In CACO-2, SCC-15 and A549 cell lines, 1 µM Les-236 did not affect KI67 protein expression. In the CACO-2 cell line, cell co-stimulation with Les-236 and rosiglitazone increased expression of KI67. In the A549 cell line, co-treatment with Les-236 and GW9662 decreased KI67 protein expression. Interestingly, in the SCC-15 cell line, co-stimulation with both, Les-236 and rosiglitazone and Les-236 and GW9662, increased KI67 protein expression. As mentioned above, in Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) toward the human cancer cell lines. The studied compound decreased ROS production in BJ, SCC-15, CACO-2, A549, and SH-SY5Y cell lines at micromolar concentrations. However, the compound increased LDH release and decreased cell metabolism/proliferation at micromolar concentrations in BJ, SCC-15, CACO-2, A549, and SH-SY5Y cell lines. Les-236 was able to increase the activity of caspase-3 in BJ (after 6 h of exposure), A549, CACO-2, and SCC-15 (after 48 h of exposure) cell lines over a wide range of concentrations (1 nM to 100 µM), which could be due to the activation of PPARγ-dependent pathways. However, the safety of this compound and its metabolites should be examined in other normal human cells. Therefore, further studies on the mechanism underlying the effects of Les-236 in vitro and in vivo are needed.In conclusion, our results showed the anticancer potential of 52+ and Mg2+, hydrocortisone, sodium pyruvate, sodium bicarbonate, dimethyl sulfoxide (DMSO), caspase-3 substrate (235400), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D6883), GW9662, rosiglitazone, hydrogen peroxide (H2O2) and resazurin (R7017) were purchased from Sigma-Aldrich . Black 96-well culture plates (655090) were purchased from Greiner Bio-One . Charcoal/dextran-treated fetal bovine serum (FBS), heat-inactivated FBS, RNA isolation kit (E0309), and fast probe real-time PCR master mix (Rox) (E0423) were purchased from EURx . F12K, MEM, and DMEM/F12 (1:1) media were purchased from Corning . The lactate dehydrogenase (LDH) cytotoxicity detection kit (11644793001) was purchased from ROCHE Applied Science . The High Capacity cDNA Reverse Transcription Kit and the TaqMan® probes corresponding to specific genes encoding β-Actin (Hs01060665_g1) and PPARγ (Hs00234592_m1) were purchased from Life Technologies . PPARγ (H1361) and KI67P (H5432) ELISA assays were obtained from Elabscience Biotechnology .Trypsin, penicillin, streptomycin, phosphate-buffered saline without CaZ-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) were described previously16  release, resazurine reduction and caspase-3 activity assays, the cells were seeded in 96-well culture plates at a density of 6 × 103 (for 6 and 24 h treatments) and 5 × 103 (for 48 h treatment) per well and initially cultured before the experiment for 24 h. For the real-time PCR assay, different cell lines were seeded onto 12-well plates and initially cultured for 24 h. For the protein measurement, different cell lines were seeded onto 6-well plates and initially cultured for 24 h. Subsequently, the medium was replaced with a fresh one by raising the concentrations  of the studied compound Les-236.Human squamous cell carcinoma (SCC-15), lung carcinoma (A549), colon adenocarcinoma (CACO-2), neuroblastoma (SH-SY5Y), and skin fibroblast (BJ) cell lines were obtained from the American Type Culture Collection . The A549 cell line was maintained in the F12K medium, SCC-15 and SH-SY5Y cell lines were maintained in the DMEM/F12 medium and BJ and CACO-2 cell lines were maintained in the MEM medium. The media used in the experiments were phenol red-free and contained 2.5 mM L-glutamine. Moreover, the media were supplemented with 10% FBS (or heat-inactivated FBS for SH-SY5Y cells) and 400 ng/mL hydrocortisone (only for SCC-15 cells). The cells were maintained in an incubator at 37 °C in a humidified atmosphere with 5% CO2DCFDA was applied. The cells were incubated with H2DCFDA in serum-free and phenol red-free medium for 45 min before treatment with increasing  concentrations of the studied compound according to a previously described method65. After incubating the cells for 3, 6, and 24 h with increasing concentrations of the compound (and 5% CO2 at 37 °C), the culture medium was replaced with a fresh one to remove extracellular residual dichlorodihydrofluorescein (DCF). Cells treated with 100 µM hydrogen peroxide (H2O2) were used as a positive control (results not shown). DCF fluorescence was measured using a microplate reader (FilterMax F5) at the maximum excitation and emission spectra of 485 and 535 nm, respectively.To determine the ability of Les-236 to induce ROS production in the cells, 5 μM H66. The assay was based on cell lysis and the release of LDH from the cytosol of the damaged cells into the surrounding medium. After 6, 24, and 48 h of treatment of the cells with increasing concentrations of the studied compound, 100 µL of the culture supernatants was collected and incubated in the reaction mixture from the kit according to the manufacturer’s protocol. After 30 min, the reaction was stopped by adding 1 N HCl and the absorbance was measured at a wavelength of 490 nm using the FilterMax F5 Multi-Mode microplate reader .The cytotoxicity detection kit is used for colorimetric assay to quantify the cell death -necrosis and secondary necrosis (after the disintegration of the apoptotic bodies in the cell culture environment)37. After 6, 24, and 48 h of treatment of cells with increasing concentrations of the Les-236 compound in 96-well culture plates, the cells in the wells were replaced by a working solution of resazurin (100 µL) and the plates were incubated at 37 °C. Fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm on the FilterMax F5 Multi-Mode microplate reader  after 30 and 60 min of dye addition.A working solution of 60 µM resazurin was prepared in a medium containing 1% FBS on the day of analysis according to a previously described method37. The lysates were incubated with caspase-3 substrate Ac-DEVD-pNA at 37 °C. Cells treated with 1 μM staurosporine were used as a positive control (results not shown). After 30 min, the absorbance of the lysates at 405 nm was measured using the FilterMax F5 Multi-Mode microplate reader. The amount of colorimetric product produced was continuously monitored for 120 min. The data were analyzed using Multi-Mode Analysis software  and were normalized to absorbance of the vehicle-treated cells.Caspase-3 activity was used as a marker of cell apoptosis. After 6, 24, and 48 h of treatment of cells with increasing concentrations of Les-236, the culture plates were frozen at −80 °C and left until assay. The frozen cells were lysed with lysis buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM DTT) at 10 °C for 10 min according to a previously described protocol20. After a 6 h exposure to 1 or 10 µM Les236, the samples were collected and total RNA was extracted from the cells using an RNA isolation kit according to the manufacturer’s instructions. The quality and quantity of the RNA were determined spectrophotometrically at 260 and 280 nm, respectively . Two-step real-time reverse transcription (RT) PCR was conducted, with both the RT reaction and the quantitative PCR (qPCR) run using the CFX Real Time System . The RT reaction was carried out at a final volume of 20 μL with 250 ng RNA (as a cDNA template) using the cDNA reverse transcription kit in accordance with the manufacturer’s instructions. Products from the RT reaction were amplified using the fast probe qPCR master mix (Rox) kit with TaqMan probes as primers for specific genes encoding ACTB and PPARγ. Amplification was carried out in a total volume of 25 μL containing 1 × fast probe qPCR master mix (Rox) and 1.0 μL of the RT product which was used as the PCR template. Standard qPCR procedures were carried out as follows: 2 min at 50 °C and 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value (Ct) for each sample was set during the exponential phase, and the ΔΔCt method was used for data analysis. ACTB was used as the reference gene.The experiment was conducted as per a previously described procedureThe levels of PPARγ and KI67 proteins were determined through ELISA after a 48 h of treatment with 1 µM Les236 or co-treatment with Les-236 and 10 µM rosiglitazone or Les-236 and GW9662. These proteins were specifically detected with ELISA and subsequently subjected to quantitative sandwich enzyme immunoassay, which was conducted according to the manufacturer’s instructions . Briefly, a 96-well plate was pre-coated with monoclonal antibodies specific to PPARγ and KI67. Standards and the collected cell extracts were added to the wells and incubated for 90 min at 37 °C. Next, the liquid was removed, and biotinylated detection antibodies (100 µL) were added to the cultures for 60 min. We washed the cells three times to remove any unbound substances, and then added horseradish peroxidase-conjugated avidin. The cells were washed again, and 90 µL of substrate solution was added to the wells for 15 min. Then, 50 µL stop solution was added to terminate the reaction and the absorbance was measured at 450 nm; the value obtained was proportional to the amount of PPARγ and KI67, respectively. The total protein concentration was determined in triplicate in each sample by using a Thermo Fisher NanoDrop device.*p < 0.05, **p < 0.01, and ***p < 0.001 vs. control cells were considered significant. Statistical significance is marked on the figures , and the values were measured in triplicate. The average of the triplicate samples was used for the statistical analyses. Considering the difference in data obtained from the measurement of fluorescence and absorbance, the results were presented as a percentage of the controls. The data were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison procedure; res Figs , 7, 8, 9res Figs , 4, 5, 6, and theLes-236 decreased the production of reactive oxygen species in BJ, SCC-15, CACO-2, A549, and SH-SY5Y cell lines.At high micromolar concentrations, Les-236 increased the release of lactate dehydrogenase in all the studied cell lines.Les-236 activated caspase-3 in A549, CACO-2, and SCC-15 cell lines at all the studied concentrations.PPARγ is involved in Les-236 mechanism of action.Supplementary data


Met and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5′ end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects.Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNA Gln, tRNASer(UCN) and ND6  and cardiomyopathies-associated tRNAIle 4269A>G and 4295A>G mutations and tRNAHis 12192G>A mutation perturbed the 3′ end processing of corresponding tRNA precursors (Leu(UUR) caused the 5′ end aberrant processing of tRNALeu(UUR) and accumulation of RNA precursors  of corresponding tRNAs, and m.4401A>G mutation at the junction of tRNAMet and tRNAGln genes  (containing 4.5 mg of glucose and 0.11 mg of pyruvate/ml), supplemented with 100 μg of BrdU/ml and 5% FBS. The mtDNA-less ρ°206 cell line, derived from 143B.TK−  of a Chinese family carrying the homoplasmic m.4401A>G mutation  and one 143B.TK−  was grow143B.TK− .The HUVECs-less-mtDNA lines were produced as detailed elsewhere ,34. TranMet and tRNAGln from the control cell line C19 and mutant cell line III-3 were sequenced after cDNA synthesis, PCR amplification, and cloning, as detailed elsewhere  and GLN1 (5′CAAAATTCTCCGTGCCACCTATCA-3′), respectively. The second strands of these cDNAs were then synthesized by using primers MET2 (5′-CCCCGAAAATGTTGGTTATACCCTT-3′), GLN2 (5′-GATTCTCAGGGATGGGTTCGATT-3′), respectively. The artificial tDNAs were then amplified by PCR, using above primers, respectively. Those resultant PCR products were cloned in the TA vector (Invitrogen), and eight clones of each control cell tDNA and III-3-1′s tDNA were analyzed by Sanger sequence.The 5′ and 3′ ends of tRNAlsewhere . First, Met corresponding to mtDNA at positions 4365 (5′) to 4469 (3′), and tRNAGln at mtDNA positions 4438 (5′) to 4329 (3′) were cloned into the pCRII-TOPO vector carrying SP6 and T7 promoters (Clontech). After HindIII digestion, the labeled RNA substrates (104 nt for tRNAMet and 109 nt for tRNAGln) were transcribed with T7 RNA polymerase, in the presence of 10 μM ATP, CTP, GTP and UTP, pH 7.5 and 10 units RNase inhibitor at 20°C. Transcripts were purified by denaturing polyacrylamide gel electrophoresis (PAGE)  and were dissolved in 1 mM EDTA. Mitochondrial RNase P was reconstituted from purified recombinant proteins MRPP1, MRPP2 and MRPP3 as described previously . Reaction products were resolved via denaturing PAGE, then electroblotted onto a nylon membrane (Roche) and hybridized with digoxigenin (DIG)-labeled oligodeoxynucleotide probes for tRNAMet precursor (5′-TAGGATGGGGTGTGATAGGTGGCACGGAGAATTTT-3′) and tRNAGln precursor (5′-AGTAAGGTCAGCTAAATAAGCTATCGGGCCCATACCC-3′). DIG-labeled probes were generated by using DIG-oligonucleotide Tailing kit (Roche). The hybridization and quantification of density in each band were performed as detailed previously , as detailed elsewhere  from intact cells or mitochondria isolated from the various cell lines , and hybridized with a set of DIG-labeled RNA probes: ND6, ND1, COX1, COX2, CYTB, 12S rRNA, 16S rRNA, and β-actin as a control, respectively. Probes were synthesized on the corresponding restriction enzyme linearized plasmid using a DIG RNA Labeling kit (Roche). The plasmids used for RNA probes were constructed by PCR-amplifying fragments of ND6 (positions 14343 to 14618), ND1 (positions 3506–3839), COX1 (positions 7146–7425), COX2 (positions 7823–8156), CYTB (positions 14824–15208), 12S rRNA (positions 1201–1235), 16S rRNA (positions 2245–2635), and β-actin  and cloning these fragments into the pCRII-TOPO vector . Quantification of density in each band was performed as described previously . Inhibitors were used at the following concentrations: Oligomycin (to inhibit the ATP synthase) (1.5 μM), Carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP)  (0.15μM), Antimycin A (to inhibit complex III) (5 μM) and Rotenone (to inhibit complex I) (1 μM).The rates of oxygen consumption (OCR) in various cell lines were determined with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience), as detailed elsewhere . Cells wMitochondrial membrane potential from various cell lines was examined with JC-10 Assay Kit-Flow Cytometry (Abcam) following general manufacturer's recommendations with some modifications, as described previously ,47.The levels of mitochondrial reactive oxygen species (ROS) generation in various cell lines were measured using MitoSOX assay as detailed elsewhere ,33,48.5 cells of each cybrid cell line were incubated with the endothelial basal medium (ScienCell) in the absence and presence of 500 nM rapamycin (inducers of autophagy) and 10 μM chloroquine  at 37°C for 18 h, and spin down and washed with PBS. The resultant samples were resuspended with CYTO-ID®-Green reagent and analyzed using a Novocyte flow cytometer (ACEA Biosciences) in FITC channel.The fluorescence-based cytometry to analyze the level of mitophagy was performed using CYTO-ID® Autophagy Detection Kit (Enzo), as detailed elsewhere ,50. In bWound healing assays were performed as detailed elsewhere ,51.A tube formation assay using growth factor-reduced Matrigel was carried out as detailed previously ,52,53.t-test contained in Microsoft Office Excel (version 2013) and GraphPad Prism . P indicates the significance, according to the t-test, of the difference between mutant and control mean. Differences were considered significant at a P < 0.05.Statistical analysis was performed by the unpaired, two-tailed Student's Met and tRNAGln, the 5′ and 3′ ends of the mitochondrial tRNAMet and tRNAGln from the control cell line C19 and mutant cell line III-3 were analyzed by Sanger sequence after cDNA synthesis, PCR amplification, and cloning, as described elsewhere , tRNAAsp and tRNAGly are located at the H-strand transcript , tRNASer(AGY) derived from the H-strand transcripts and 8 tRNAs including tRNAGln, derived from the L-strand transcripts , respectively. As shown in Figure Gln and tRNAMet in three mutant cybrids were 41% and 55% (P < 0.01) of the mean values of three control cybrids, respectively. Strikingly, the average steady-state levels of other 7 tRNAs from the L-strand transcript were significantly decreased. Especially, the average levels of tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer(UCN), tRNAGlu and tRNAPro in three mutant cybrids were 42%, 33%, 38%, 55%, 52%, 67% and 46% of those in three control cybrids, respectively. Interestingly, the average levels of 8 tRNAs from L-strand transcript in the mutant cell lines were 46.75% of those in the control cell lines. In contrast, the steady-state levels of other 13 tRNAs, such as tRNALys, tRNALeu(UUR) and tRNASer(AGY) from H-strand transcript in the mutant cybrids were comparable with those in three control cybrids. Furthermore, these results were verified by tRNA Northern blot analysis using mutant and control immortalized lymphoblastoid cell lines . These data indicated that the m.4401A>G mutation led to the metabolic defects of all 8 tRNAs, derived from L-strand transcript and tRNAMet from H-strand transcript.Among 22 mitochondrial tRNAs, 8 tRNAs such as tRNAanscript . It was β-actin as a control, respectively. These experiments using ND6 probe revealed the presence of RNA species with ∼1.1 and ∼4.3 kb long, respectively, as shown in Figure P < 0.01)  and ND1]  Figure . Howeverand ND1] ,11,27 frb) and ATP8 (subunit 8 of H+-ATPase)] in mutant and control cybrid cell lines with a nuclear encoding mitochondrial protein Tom20 as loading control. As shown in Figure P <0.01), relative to the mean value measured in three control cybrid cell lines , CO2 (subunit II of cytochrome c oxidase); CYTB  encoded by nuclear genes. As shown in Figure We then examined the levels of five subunits of OXPHOS complexes in control and mutant cybrids by Western blot analysis using the total OXPHOS human antibodies cocktail containing antibodies for mtDNA encoded subunit CO2 of cytochrome c oxidase and four other polypeptides  of the mean values measured in three control cybrid cell lines. To further investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cybrid cell lines, OCR was measured after the sequential addition of oligomycin, FCCP, antimycin A and rotenone (P < 0.01), 86% (P = 0.42), 36% (P < 0.01), 32% (P < 0.01) and 61% (P < 0.01) relative to the mean value measured in the control cybrid cell lines, respectively.To evaluate if the impairment of translation caused by the m.4401A>G mutation affects oxidative phosphorylation, we measured the oxygen consumption rate (OCR) of various mutant and control cybrid cell lines using a Seahorse Bioscience XF-96 Extracellular Flux Analyzer ,46. As srotenone ,57. The P < 0.01) of the mean value measured in three control cybrids. Conversely, the levels of ΔΨm in three mutant cybrids in the presence of FCCP were comparable with those measured in three control cybrid cell lines.The mitochondrial membrane potential (ΔΨm) generated by proton pumps  is the central bioenergetic parameter that controls respiratory rate, ATP synthesis and the generation of reactive oxygen species . The mit2O2 stimulation (P < 0.01) of the mean value measured in control cybrid cell lines under unstimulated conditions. As illustrated in Figure P < 0.01) of the mean values measured in three control cybrid cell lines under stimulation conditions.Excess productions of ROS caused by mitochondrial dysfunctions have been linked to the pathology of hypertension ,59. To amulation ,48. Geom® Autophagy Detection Kits were used with flow cytometry to examine the degree of autophagy of mutant and control cell lines (P < 0.01) of the mean values measured in three control cybrid cell lines  of the mean values measured in three control cell lines lacking the mutation. However, the levels of p62 in mutant cell lines carrying the m.4401A>G mutation were comparable with those in three control cell lines lacking the mutation , compared with the average values of three control cybrid lines.To examine the effect of m.4401A>G mutation-induced alterations on angiogenesis and wound regeneration, the wound healing assays were carried out in various mutant and control cybrids with live-cell microscopy ,51,63. FThe effect of m.4401A>G mutation on angiogenesis was further evaluated in various mutant and control cell lines by the tube formation assay ,53. The Met at the H-strand transcript and tRNAGln at the L-strand transcript , respectively , 12S rRNA, 16S rRNA, mRNAs and their precursors between the mutant and control cell lines. The processing of the precursor H-strand transcripts, excised by RNase P and RNase Z, referred to as ‘the tRNA punctuation model’, may undergo simultaneously as the transcriptions of HSP2 proceeds (Met 5′ end processing caused by the m.4401A>G mutation did not affect the processing of other 13 tRNAs including tRNALeu(UUR), tRNALys, 12 mRNAs such as ND1 and COXII, 12S rRNA and 16S rRNA which are co-transcribed from the L-strand mtDNA , tRNAGlu, and tRNAPro and the accumulation of longer and uncleaved precursors in the mutant cybrids, as compared with those in control cybrids. In fact, the deafness-associated m.7445A>G mutation in a spacer immediately to the 3′ end of tRNASer(UCN) not only altered the 3′ end processing of tRNASer(UCN) precursor but also had long-range effects on ND6 expression , ATP-linked OCR (66%), maximal OCR (64%) and reserve capacity (68%), in contrast with 20–23% reductions in the rates of total O2 consumption, complex I, complex III and complex IV-derived O2 consumption observed in the lymphoblastoid cell lines derived from a Chinese family carrying the m.4401A>G mutation (Met and tRNAGln in the mutant HUVEC-derived cybrids, and ∼30% reductions in the steady state levels of these tRNAs in the mutant lymphoblastoid cell lines (The shortages of tRNAmutation . Strikinll lines . Dysfuncll lines ,72,73. Tll lines ,58,74,75ll lines . Alteratll lines ,82. In tll lines , in contll lines . These iThe m.4401A>G mutation-induced mitochondrial dysfunctions, including decreased respiration, ATP production, and increased production of ROS led to the profound impacts on the vital functions of endothelial cells ,84–87. IMet and tRNAGln precursors. The m.4401A>G mutation yielded the low level of tRNAMet but did not affect the levels of other 13 tRNAs, 12 mRNAs 12S rRNA and 16S rRNA from the H-strand transcript. Conversely, the m.4401A>G mutation led to the significant decreases in the levels of all 8 tRNAs and ND6 but the accumulation of longer and uncleaved precursors from the L-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects resulted in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects.In summary, our findings demonstrated the pathogenic mechanism leading to an impaired oxidative phosphorylation in HUVECs-derived cybrid cell lines carrying the hypertension-associated m.4401A>G mutation. The m.4401A>G mutation perturbed the 5′ end processing of tRNAgkz742_Supplemental_FileClick here for additional data file.


Citrullus colocynthis leaves together with four known cucurbitacins. Their structures were established on the basis of their spectroscopic data (mainly NMR and mass spectrometry). Evaluation of the in vitro cytotoxic activity of the isolated compounds against two human colon cancer cell lines (HT29 and Caco-2) and one normal rat intestine epithelial cell line (IEC6), revealed that one of the isolated compounds presented interesting specific cytotoxic activity towards colorectal cell lines.Two new tetracyclic cucurbitane-type triterpene glycosides were isolated from an ethyl acetate extract of  Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) is a valuable plant widely distributed in the desert areas of the world including Tunisia + consistent with the molecular formula C47H60O15 (∆ppm = −0.11)   and a downfield δC around 79–80 for the C-25 esterified derivatives + .25-p-Coumaroyl-3′-acetyl-2-O-β-d-glucocucurbitacin I (2). Yellow amorphous powder; c 0.0005, MeOH), UV (MeOH) λmax (nm): 229, 312; IR cm−1: 3406, 2983, 2919, 1705, 1684, 1633, 1615, 1079; 1H-NMR  and 13C-NMR  spectral data see m/z 887.3823 [M + Na]+ .2 until reaching 90% confluence and medium was changed every 2 days. Cells were used for experiments before the 4th passage after thawing (culture duration = 1 month). Forty-eight hours before addition of compounds, cells were plated in a 96-well plate  with DMEM and 10% FCS or embedded in Matrigel for 3D culture. Spheroids of HT29, Caco2 or IEC6 were obtained from 1 × 104 cells embedded in 4 µL Matrigel seeded on top of 20 µL polymerized Matrigel in 48-well plates. After 30 min, DMEM with 10% FCS was added. Spheroids were observed daily using an inverted microscope (Apotome)  to follow their growth. Forty-eight hours after seeding, spheroids showed round structures. Compounds 1–6 were dissolved in EtOH 10% to obtain an initial concentration of 10 mg/mL. Then, the compounds were incubated with cells  in DMEM with 5% FCS at increasing concentrations of 1, 10, 50 and 100 µg/mL for 48 h. After, the cells were incubated with MTS-tetrazolium compound  for 4 h and the colorimetric analysis of surviving cells was made by spectrophotometry . The cytotoxicity of compounds 1–6 was then estimated by comparing the variation of cell survival between treated and control assays (EtOH 10%) and was expressed as percentage.HT29 (ATCC-HTB-38) and Caco-2  cells from human colorectal cancer, and IEC6 (ATCC-CRL-1592) cells from rat small intestine, were from ATCC  and were cultured in Dulbecco’s modified Eagle’s medium (DMEM Cat. No. 31966 with Glutamax and 1 mM sodium pyruvate) supplemented with 100 U/mL penicillin/streptomycin and 10% fetal calf serum (FCS) without complement. Caco-2 culture medium was supplemented by 1% non-essential amino acids. All cell culture reagents were from Invitrogen . The Caco-2 cell line was obtained from a 72-year-old male patient and exhibits APC mutation but wild-type BRAF. The HT29 cell line comes from a 44-year-old female patient and exhibits both APC and BRAF mutations. All cell lines were cultured at 37 °C and 5% CO3CN (+0.1% formic acid) over 30 min at a flow rate of 460 µL/min. The QTOF-MS was operated both in NI and PI mode at a resolution of approximately 10,000 . The data were acquired over an m/z range of 50–1000 in full scan mode. The capillary and cone voltages were set to 2.5 kV and 40 V, respectively. The source temperature was maintained at 120 °C, and the desolvation and cone gas flows were set to 900 L/h at 350 °C and 20 L/h, respectively.LC-MS profiles of crude extracts were acquired using a UHPLC-QTOF-MS equipped with an ESI source . Plant extracts were separated using a UPLC BEH C18 Acquity column  with a gradient from 95% water (+0.1% formic acid) to 95% CHCitrullus colocynthis leaf ethyl acetate extract resulted in the isolation of two new cucurbitacins named 6′-acetyl-2-O-β-d-glucocucurbitacin E (1) and 25-p-coumaroyl-3′-acetyl-2-O-β-d-glucocucurbitacin I (2) along with four known other cucurbitane derivatives (3–6). The new coumaroyl cucurbitacin derivative 2 displayed interesting specific cytotoxic activity towards colorectal cell lines. Nonetheless, these preliminary results should be confirmed on other normal and cancer cell lines and on ex vivo and in vivo cancerous tissues, before considering further development for these compounds.The chemical investigation of


A novel antiproliferative molecular scaffold was designed by rational modification of known antiandrogens, achieving a significant improvement in anti-cancer activity. 50 values in the low μM range. The cytotoxicity profile was also evaluated for the novel structures in the HEK293 cell line.Prostate cancer is a major cause of male death worldwide and the identification of new efficient treatments is constantly needed. Different non-steroidal androgen receptor antagonists are approved also in the case of castration-resistant cancer forms. Using a rational approach and molecular modelling studies to modify the structure of antiandrogen drug bicalutamide, a new series of phenylsulfonyl-benzamide derivatives was designed and synthesised. Their antiproliferative activities were evaluated in four different human prostate cancer cell lines and several new compounds showed significantly improved IC Unfortulutamide  are two 1, ring B can bend to occupy an inner portion of the ligand-binding domain, thus allowing the closure of the receptor into its agonist conformation. Treatment with enzalutamide induces instead a F876L mutation in the AR, which also confers an antagonist to agonist switch in activity for the drug.7One of the most common mutations found for bicalutamide is W741L in helix 12 of the receptor,4), derived from the natural pigment curcumin, has been reported, in which a central phenyl group is acting as linker connecting two different aromatic rings.9Most non-steroidal antiandrogens are structurally characterised by two differently substituted aromatic rings, named ring A and ring B, connected by a linker, either linear  or cyclic  .8 Recent2 substituent), replacing the classical 4-cyano substituent. The phenolic 4-hydroxy group of 4, previously reported as essential for AR antagonistic activity,With the aim to rigidify the structure of bicalutamide and obtain new anticancer agents, we designed a novel molecular scaffold in which we replaced the flexible bicalutamide methyl-hydroxy-methylene linker with a rigid phenyl group, maintaining the two lateral aromatic rings commonly found in antiandrogen compounds . MoreoveFollowing this approach, a novel family of phenylsulfonyl-benzamides was developed , and the13a, which maintains bicalutamide and enzalutamide aromatic substituents in ring A and is characterised by the fully oxidised sulfonyl linker, was chosen as representative for the novel series of compounds. In a previous work,13a was performed using MOE2015.10,13a aligns well with the binding pose of the parent compounds, with the newly introduced phenyl ring conferring the desired structural rigidity to lock the molecule in the active conformation  similarity score obtained for the alignment with enzalutamide is a further confirmation that the introduction of the phenyl linker rigidifies the novel structure, potentially locking the molecule in an active conformation.A series of flexible alignment and molecular docking studies was performed to evaluate the proposed structural modifications. For these analyses, compound ormation . Moreove13a in the ligand binding domain (LBD) of the open AR homology model, obtained using Plants docking software.13a in the bicalutamide-resistant AR mutant W741L crystal structure ,13a and helix 12 . These two sulfoxides were characterised by low solubility in the reaction solvent, therefore complete oxidation to the corresponding sulfone was much slower and did not go to completion within the reaction time. Using this synthetic approach, 24 novel phenylsulfonyl-benzamides derivatives were prepared, purified and fully characterised.In the first step a copper catalysed C–S coupling reaction was used for the preparation of intermediates Standard bicalutamide and enzalutamide were also prepared following reported procedures.1050 in μM), along with the overall effect in the four cell lines reported as geometric mean. Activity found for bicalutamide and enzalutamide is consistent with previously published data for some of these specific cell lines,All new compounds along with the two standards were tested for their ability to inhibit survival and/or cell proliferation in four human prostate cancer cell lines: LNCaP, 22Rv1, VCaP, androgen-sensitive cell lines, and DU145, a non hormone-sensitive cell line. 50 values ranging from 3.2 μM to >100 μM. Compared with standard enzalutamide, antiproliferative effect was improved up to 8-fold.Considering their overall profile in the four cell lines (geometric mean), almost all the new derivatives performed significantly better than bicalutamide, improving its effect up to 16-fold. The new inhibitors showed concentration-dependent activity with mean IC1 in ring B does not seem to influence the anticancer profile of the scaffold, as no substantial differences were found between the OH group in the para position or the CF3 in meta (i.e.11avs.12a). This finding is in accordance with the proposed binding mode for 13a, in which the R1 substituent is facing outside the AR binding domain without making specific interactions.Substituent R2), with a decrease of effect in the sulfones up to 8-fold. In the new scaffold presented in this work, no particular differences can be observed between thioether and sulfone compounds, with retained activity in most sulfones (i.e.11avs.13a) and only a small increase of IC50 values up to 2-fold in few cases (i.e.11cvs.13c). The partially oxidized derivatives (X = SO) retain the effect of the corresponding thioethers and sulfones (i.e.11e and 13evs.14e).In both our previous work on bicalutamide derivatives and in another published work on analogues of SARMs (selective androgen receptor modulators),2 group in the para position of ring B is associated with retained activity, as found for mono 3-CF3 derivative 11h. Moving the trifluoromethyl group from meta to para position does not have a significant effect on activity (i.e.11hvs.14g), whereas the ortho substitution is characterized by a 3-fold reduction of overall effect in the four cell lines (i.e.11hvs.11i). Replacing the trifluoromethyl group with a bigger –SF5 moiety in para or meta position is associated with a mildly improved activity profile (i.e.11gvs.14e).Removal of the CN or the NO3 in meta , gives the best results in terms of overall activity in the four cell lines (i.e.11a and 13hvs.14c), as we had similarly observed in our previous research efforts.meta to ortho position is associated with activity reduction (i.e.11cvs.11d).Addition of an extra CF50 value (14e). These data suggest that the newly prepared compounds are likely to retain an antagonistic effect on the androgen receptor. An interesting activity profile was also found in the DU-145 cell line (the least sensitive to our new molecules), which expresses low levels of the androgen receptor50 values across the four cell lines), suggesting that besides its canonical anti-androgen receptor action, a different antiproliferative mechanism could also be involved. The new phenylsulfonyl-benzamides, in addition to potentially increase the antagonistic activity on the AR, might also enhance the potential off target effect, even though in some cases they seem more selective for androgen-sensitive cell lines than bicalutamide . As a general observation, the improved antiproliferative activity of the novel compounds should be regarded as the result of the whole structure of each single derivative, since the data found appear to be a combination of the simultaneous effects of the substituent in ring B and linker X.Examining each individual cell line, most of the new compounds showed the best results in the cell lines that highly express the androgen receptor. Among them, LNCaP cells were found to be the most sensitive to the new derivatives, with different compounds active at low μM concentrations and one reaching a sub μM IC11d, 11e and 13e) significantly reduced cell viability if compared to bicalutamide , and only one of them (13e) killed more than 50% of cells. Due to their higher cytotoxicity in comparison with bicalutamide, the antiproliferative effect of these three compounds in the prostate cancer cell lines could also be a consequence of their intrinsic toxicity, besides the canonical AR antagonist activity. Comparing the rest of the compounds with bicalutamide, even if some of them are characterized by an increased cytotoxicity, cell viability reduction is not statistically significant and less than 40% in most cases, therefore the observed cytotoxicity is comparable to the one found for bicalutamide. Moreover, it is worth to note that bicalutamide reaches its antiproliferative IC50 at 50 μM, therefore it would not show reduction of cell viability at 10 μM. On the contrary, most of our new compounds showed antiproliferative activity in the four prostate cancer cell lines at a low μM concentration (<10 μM), and even if some of them show a reduction of cell viability by 35–40%, their anticancer therapeutic window is still wide enough for consideration as a potential treatment for prostate cancer. In particular, compounds 11h, 12h, 13b and 13h are associated with a promising antiproliferative/cytotoxicity profile, with an antiproliferative effect much better than bicalutamide (<16 μM), and with the same cytotoxicity of bicalutamide at 10 μM .A Cell Titer Blue viability assay was performed in the human embryonic kidney cell line HEK293, in order to evaluate the cytotoxicity profile of the newly prepared compounds. 11h, 12h, 13b and 13h have been selected as candidates for further investigations, due to their improved anticancer properties and their cytotoxicity profiles. With the aim to identify a pre-clinical candidate, these compounds will be studied for their binding to the AR, their potential off target effect and their pharmacokinetic properties.A novel family of phenylsulfonyl-benzamides was designed and synthesised after rational modification of the structure of antiandrogen drug bicalutamide, and many of the novel derivatives prepared show improved anti-prostate cancer properties. In particular, most of the new compounds display significantly improved anticancer activities in four different prostate cancer cell lines, with a cell viability reduction effect comparable to the one found for bicalutamide. Differently from previously reported SARMs, no significant difference in antiproliferative activity has been observed between our novel thioether and sulfone derivatives, representing a promising feature for future metabolic stability considerations. Within the 24 novel analogues evaluated, 1H, 13C, 19F) and LRMS. All final compounds were found to be >95% pure by HPLC.All chemistry, biology and molecular modelling experimental procedures, along with compound characterisation, are fully described in the ESI.Supplementary informationClick here for additional data file.