Chlorinated compounds in a water environment pose serious threats to humanity. A nanoscale zerovalent iron (nZVI) has desirable properties for water dichlorination, but its reactivity is still limited by agglomeration and oxidation. In this study, the mesoporous carbon (MC) derived from biomass waste was prepared for immobilizing nZVI, and the nZVI@MC was further modified by sulfur (S-nZVI@MC) to relieve surface oxidation. The synergistic effect between nZVI and surface modification, the reaction conditions and the removal mechanism were investigated systematically. The characterization results showed nZVI was successfully loaded on the surface of MC, and the aggregation of nZVI was prevented. Moreover, sulfidation modification resulted in the formation of FeS on the surface of nZVI, which effectively alleviated surface oxidation of nZVI and promoted the electron transfer. Batch experiments demonstrated S-nZVI@MC had greatly enhanced reactivity towards 2,4,6-trichlorphenol (TCP) as compared to MC and nZVI, and the removal rate could reach 100%, which was mainly attributed to the significant synergistic effect of MC immobilization and sulfidation modification. Furthermore, the TCP removal process was well described by a Langmuir adsorption model and pseudo-second-order model. The possible mechanism for enhanced removal of TCP is the fast adsorption onto S-nZVI@MC and effective reduction by S-nZVI. Therefore, with excellent reducing activity and antioxidation, S-nZVI@MC has the potential as a pollutant treatment. Nowadays, the scarcity of water resources and increasing water contamination are a worldwide environmental problem that have drawn extensive attention. Chlorophenols are a class of typical organic contaminants that have been utilized widely in various industrial and agricultural fields. Specifically, 2,4,6-trichlorphenol (TCP) has been listed among United States Environmental Protection Agency (US EPA) priority pollutants due to its high toxicity, mutagenicity, and carcinogenicity ,2. VarioIn the past two decades, nanoscale zerovalent iron (nZVI) has been demonstrated to be an effective solution for the degradation/elimination of various pollutants including chlorinated organic compounds, heavy metal ions and antibiotics due to its strong reductive capability, low cost and environmental friendliness ,7,8. HowImmobilizing nZVI particles on porous material supports has been demonstrated as a tactful way to obtain high-efficiency and stable nZVI . On the 2 and other coexisting solutes, which would impede electron transfer from nZVI core to target contaminates [It should be noted that although supported nZVI can prevent the agglomeration and improve the stability of nZVI, it is easily oxidized by water, Oaminates . In viewaminates ,19,20. Oaminates . He et aaminates .4 in the presence of dithionite. The aggregation and surface oxidation of nZVI has been effectively alleviated due to the synergistic effect of loading and sulfidation, which is beneficial to improve its reactivity and life. The S-nZVI@MC was further used for TCP removal from water and the effects of solution pH, TCP initial concentration, S-nZVI@MC dosage were examined. The finding of this study paves the way to developing high-performance and low-cost nZVI for dichlorination and reduction of other pollutants.To take advantage of the recent development in nZVI, a sulfidized nZVI supported on MC (S-nZVI@MC) was prepared and used to remove TCP from aqueous solutions. As seen from −1) was used throughout the experiment.Materials: wheat straw was obtained from Yancheng, Jiangsu, P. R. of China. To remove impurities, the wheat straw is fully washed with deionized water and anhydrous ethanol before use. 2,4,6-Trichlorophenol  was purchased from Aladding Chemistry Co. Ltd., Shanghai, China. Hydrofluoric acid  was obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Milli-Q water , and then stirred for 15 min. The above solution was transferred into the Teflon vessel and reacted for 12 h at 180 °C. After cooling to room temperature, the product was washed by deionized water until reaching a pH of 7, and then dried at 80 °C in a conventional oven. Finally, the per-carbonized wheat straw was obtained.Per-carbonization of the wheat straw: in typical synthesis, wheat straw (3.0 g) was added into the H2 atmosphere with the ramp rate of 3 °C/min. After HF etching and water wash, the silica can be removed and MC was finally formed.Synthesis of MC: typically, the per-carbonized wheat straw was soaked in 1.0 wt % colloidal silica aqueous solution, and mass ratio of silica/straw was 3:10. The above solution was evaporated at 85 °C through continuous stirring until becoming a viscous solution. After drying at 80 °C in a conventional oven, a solid powder was formed. To obtain the MC, the precursor was calcinated at 800 °C for 2 h under a N2 inlet; 1.15 g of FeSO4·7H2O was then added to the three-neck flask, and after being stirred for about 20 min in N2 atmosphere, 100 mL containing 7.6 g sodium borohydride and different amounts of Na2S2O6  were added in a dropwise manner to the above FeSO4 solution. After the injection, stirring continued for 30 min to ensure adequate reaction. S-nZVI@MC were harvested by centrifugation and washed with ethanol three times and then dried in a vacuum. The obtained samples were carefully stored in the glove box filled with inert gas.Synthesis of S-nZVI@MC: 0.2 g MC was firstly added into 100 mL DI and ethanol mixture solution (V:V = 7:3). After sonicating for 60–120 min, the above mixture solution was poured into a 250 mL three-necked bottom flask equipped with a mechanical stirrer, and a thermometer with a temperature controller and a NThe detailed morphological information of the samples was further analyzed by transmission electron microscopy  and scanning electron microscopy  tests. Before the TEM testing, the samples were dispersed in ethanol via ultrasonication for 15 min, and then deposited a few drops onto a carbon-coated copper grid. X-ray diffraction  measurements were recorded by the X-ray diffractometerusing Cu Kα radiation . X-ray photoelectron spectroscopy  was tested on a Perkin-Elmer PHI 5000C ESCA  system with a dual X-ray source, using the 45 MgKα (1253.6 eV) anode and a hemispherical energy analyser. Before all tests, the samples were sealed into sample tubes in a glove box, and then shipped to the test instrument.−1) and solution pH (4–11) were further investigated. The solution pH was adjusted by 0.1 M HCl or NaOH solution. The solid-liquid phases were taken out after shaking for a predetermined time, and followed by filtration using 0.22 μm cellulose nitrate membrane filter. The concentration of TCP was measured with an ultraviolet–visible spectrophotometer . The removal efficiency was calculated according to the following equations:0 and Ct (mg L−1) are the initial and final concentrations of TCP in the feed solution, respectively.Batch adsorption experiments were conducted by shaking a mixture of obtained S-nZVI@MC with 100 mL TCP aqueous solution of certain concentration in a series of 150 mL plastic bottles. Such adsorption experiment parameters as contact time (0–180 min), initial concentration of TCP  on the XRD pattern of S-nZVI@MC, which may be due to the low sulfur content or poor crystallinity of sulfur species [2O3 was observed, indicating that only part of the nZVI reacted with TCP. In addition, the reaction between such non-target reactants as H2O and nZVI should be effectively inhibited, which is attributed to the protection effect of FeS [ species . After 3t of FeS .2−, Sn− and SO42−, respectively [2−, Sn− and SO42− is 27.45%, 48.30% and 24.24%. After the reaction with TCP, the peak intensity of S2− decreased obviously, accompany with the increase of Sn−  and Fe (III), respectively [2p1/2 for Fe (III). Besides, the satellite peak positions for Fe2p1/2 and Fe2p3/2 were 718 and 733.4 eV. Before reaction, two broad peaks at 724.8 eV (Fe2p1/2) and 711 eV (Fe2p2/3) corresponding to iron oxides (Fe2+ and Fe3+) are obvious  is strengthened, while the peak corresponding to Fe (II) is weakened  was the removal capacity of TCP at time t (min) and equilibrium, respectively. k1 (min−1) and k2 (g mg−1 min−1) was the equilibrium rate constant of both kinetic models [The adsorption kinetics of MC, nZVI and S-nZVI@MC were further investigated as the following equations:c models ,29.2, the pseudo-second kinetic model can better describe the TCP removal behavior, suggesting that the TCP removal by nZVI and S-nZVI@MC is controlled by chemical processes, and the electronic exchange existed between TCP and S-nZVI@MC. Obviously, the rate constant k2 of S-nZVI@MC was 0.0018 g mg−1 min−1, higher than that of MC and nZVI (0.0012 and 0.0007 g mg−1 min−1), which further reveals a higher affinity between S-nZVI@MC and TCP owing to the hydrophobic effect [k of S-nZVI@MC further indicates that the sulfide treatment can increase the reactivity.The kinetic fitting results were shown in c effect . The hig2- results in ineffective protection of nZVI, and thus the removal efficiency of TCP is lower. The removal efficiency decreased when the S/Fe further increased to 0.25, because excessive iron sulfide, especially FeS2 with a higher band gap (0.95) generated could slow down electron transfer [Fe/S is one of the most critical factors that would influence the reactivity of S-nZVI@MC. The S-nZVI@MC particles sysnthesized at different S/Fe were further used for TCP removal, and the results are presented in transfer . MoreoveAs known, the pH value of aqueous solution has great effect on nZVI. In acidic conditions, nZVI could be rapidly corroded and tends to exhibit better activity. While in alkaline conditions, a large hydroxide passivation layer is formed on the surface of zero-valent iron, thereby inhibiting its activity. It has been demonstrated that sulfidation modification can promote electron transfer and inhibits surface passivation. However, the effect of sulfidation on nZVI reactivity under different pH conditions still needs further discussion. Herein, the removal efficiency of S-nZVI@MC under different pH value ranging from 4 to 11 was investigated. As shown in −1 TCP aqueous solution and the results are shown in −1, respectively. When the dosage is high, there are more surface reactive and adsorptive sites in the reaction system, so the removal rate of TCP is higher. However, an excessive dosage is likely to cause agglomeration of materials and low utilization of reaction sites. Hence, the dosage of the further study is 0.5 g L−1 for TCP removal.The effect of S-nZVI@MC dosage was further investigated under the condition of pH = 4.0, 30 mg L−1. Generally, the TCP removal efficiency is decreased when the initial concentration increased. As the initial concentration increased from 30 to 100 mg L−1, the removal efficiency decreased from 91% to 32%. With higher initial concentration, the surface of S-nZVI@MC is easier to passivate, and thus the diffusion of contaminants and the electron transfer between nZVI and contaminants are inhibited, resulting in a reduced contaminants removal efficiency. Besides, at high concentrations, a large number of target contaminant molecules compete for limited reaction sites on the surface of the S-nZVI@MC, which also leads to reduced TCP removal efficiency [2 (0.993). In contrast, the Freundlich model has a relative lower R2 (0.936). Hence, the adsorption of TCP on the surface of S-nZVI@MC has been dominated by monolayer adsorption [ficiency . The ads−1 TCP solution with the dose of 0.5 g L−1 and reaction time of 3 h. As shown in Reusability is another critical property for the application of nZVI-based material. The reusability of S-nZVI@MC was further tested in 30 mg LIn summary, biomass-derived MC-loaded S-nZVI was successfully prepared for effective TCP removal. MC was selected as the support for nZVI, because its mesoporous structure was beneficial to nZVI dispersion and mass transfer. Moreover, MC prepared from biomass waste was a cost-effective material for practical application. Further surface sulfidation improved the antioxidation of nZVI due to the protection effect of FeS shell. SEM, TEM, XRD and XPS measurements demonstrated that the nZVI particles were successfully loaded on the MC supporter and the FeS was well coated on the surface the nZVI. Furthermore, the aggregation of nZVI and the iron oxide formation was prevented according the structure characterization. S-nZVI@MC was most effective for TCP removal as compared to MC and nZVI, and the removal efficiency reached the highest value at a ratio of S/Fe = 0.125. The effect of ambient conditions such as solution pH, initial concentration and dosage on TCP removal were also studied, and results revealed that the low solution pH value and concentration and higher dosage had positive effect on TCP removal. Furthermore, the removal behavior was fitted with a Langmuir model and pseudo-second kinetic model. The removal mechanism of S-nZVI@MC towards TCP could by analyzed by fast adsorption and effective reduction by S-nZVI. Therefore, the synergistic effect of MC loading and surface sulfide-modification promoted high removal efficiency of S-nZVI@MC towards TCP.


Background: Prunusarmeniaca is a member of the Rosacea family. The most important ingredient of this family is amygdalin that is believed to have anti-tumor and analgesic properties. The aim of this study was to evaluate the anti-proliferative effects of Armeniacae semen extract on the acute leukemia, NALM-6, and KG-1 cell lines, and investigate the effect of the extract on apoptosis of these cell lines and caspase-3 gene expression. Materials and Methods: We prepared aqueous, ethyl acetate, and hydro alcoholic extracts of the Armeniacae semen. The NALM-6 and KG-1 cell lines and mononuclear cells (PBMCs) of healthy controls were treated with different doses of the extracts for 48 hours, and then cell viability was investigated with the MTT test. High-Performance Liquid Chromatography was done for amygdalin identification. The percentage of apoptotic cells was determined using the Annexin V-FITC/PI flow cytometric kit, and caspase-3 gene expression was evaluated.Results: MTT test revealed that the strongest Inhibition Concentration (IC50) in KG-1 and NALM-6 cell lines was related to the ethyl acetate extract. This extract did not have toxic effects on PBMCs. Flow cytometric analysis showed that the ethyl acetate extract at its IC50 concentration led to almost 50% apoptosis in both cell lines after 48 hours. In the molecular examination, after treatment, a significant increase was seen in caspase-3 gene expression in NALM6 and KG1 cells compared to the control .Conclusion: Our data confirmed that the ethyl acetate extract of Prunusarmeniaca could reduce the proliferation of KG-1 and NALM-6 cell lines probably by activating the apoptotic pathway. Based on the type  Acute leukemia refers to rapid and clonal proliferation of lymphoid and myeloid progenitor cells in the bone marrow  7,8. of stem cell involved, it is divided into two major groups: AML (acute myeloid leukemia) and ALL (acute lymphoid leukemia)   10 . Rosaceous plants, which are widely distributed, produce different economically important products, including many edible fruits, as bitter almonds, apricots, peaches, plums, etc   11 . Hee-Young Kwon et al. (2003) showed that Persicae semen extract induces apoptosis in human promyelocytic leukemia (HL-60) cells12. Jasmina Makarevic et al. (2014) reported that amygdalin from apricot kernels affects bladder cancer cell adhesion and invasion in vitro   13 . Because of these features and the lack of coherent studies on various types of leukemia, we decided to use the Armeniacae semen, a member of the Rosacea family, which contains large amount of the amygdalin, to evaluate its anti-proliferative effect on the acute leukemia, NALM-6 (ALL) and KG-1 (AML) cell lines. In addition, we investigated the effect of the Armeniacae semen on apoptosis of these cell lines and caspase-3 gene expression.Amygdalin has an antitumor effect by settling carcinogens in the body, inhibiting the nutritional source of cancer cells, and blocking the growth of the tumor cells. It can also improve the symptoms of patients in the last stages of cancer and increase their survivalCell culture◦C with 5% CO2 and 95% humidity. The medium was changed every 2-3 days.NALM-6 and KG-1 acute leukemia cell lines , which were provided by the Pastor Institute of Iran, were grown and sub cultured in RPMI1640 containing 20mM HEPES-buffer and glutamax 1%  supplemented with 10% heat-inactivated FBS   and 100µg/ml penicillin/streptomycin (Biosera). Mononuclear cells were isolated from the peripheral blood of healthy individuals using Ficoll-Paque. The cultures were incubated at 37Extracts preparationTwo hundred grams of the Armeniacae semen was hatched from the shell and dried in the shade for a week. The seeds were then crushed by a pounder. At first, the dry powder was macerated in a petroleum ether-solvent to remove oils to not disturb the cytotoxic test. After oil extraction, the seed powder was allowed to dry completely and the solvent evaporate. Then, 50 g of the powder was weighed to prepare an aqueous extract. For this purpose, the seed powder was macerated in 90 ° C water for 30-45 minutes. Then, 500 ml of ethyl acetate, was added to the rest of the powder. After 48 hours, the solvent was removed and a new ethyl acetate solvent was added again. The procedure was repeated 3 times. The above processes were also repeated for 60% methanol. The extracts from each step were filtered with filter papers, transferred to a rotary balloon  and concentrated at 100 rpm at 40 ° C.MTT assay4 cells/well. Both cell lines were treated with aqueous, methanol, and ethyl acetate extracts of the Armeniacae semen at concentrations of 0.125, 0.25, 0.5, 1 mg/ml for 48h. PBMCs were treated with the extract that had the best IC50 on the MTT test at concentrations of 0.125, 0.25, 0.5, 1 mg/ml for 48h. After this time, 50µl MTT  was added to each well and incubated for 4h. Then, the supernatant was removed and 100μl DMSO  was added to dissolve MTT. Following incubation for 15 minutes, the OD was read with an ELISA plate reader  at 492 nm. The assay was performed at least three times.To check cell viability, we used the 3--2, 5 di-phenyl tetrazolium bromide (MTT) assay. The cells were seeded in a 96-well plate at a concentration of 5×10SPE methodA solid-phase extraction cartridge (Chromafix-Germany) was used to separate and concentrate the target material of the extract, including a mixture of different materials. At first, the cartridge was washed with 5 ml of methanol and activated with 5ml of water. Then, 5 ml of the extract at a concentration of 20 mg/ml was passed through the cartridge. After washing with 10% methanol aqueous solution, to remove co-adsorbents, the remaining analytes were collected on SPE with 5 ml of pure methanol.Amygdalin identification by reverse phase HPLC18 column (250×4.6mm). The column temperature was 30°C, and the detection wavelength was set at 215 nm. At first, the amygdalin standard was injected to the Agilent 1260 chromatographic system, including a UV detector and ChemStation data system at a concentration of 100μg / ml and a volume of 20 μl, and then the exact location of the peak was determined. This action was repeated 3 times. Then, 20 μl of the specimen (isolated by SPE) was injected and after peak observation, the test was repeated 3 times. By comparing the peak area of the standard and sample, the percentage of amygdalin in the extract was obtained.In order to Amygdalin identification in the extract that had the best IC50 on the MTT test, the gradient method was used to elute other components quickly and reduce the analytical time after amygdalin peak observation. For this purpose, the mobile phase was adjusted to 15: 85 methanol-water for 30 minutes and pure methanol after 30 minutes. The mobile phase was filtered before use with a vacuum filter system containing a 0.45μm filter (Millipore-Germany). Separation was conducted on a CFlow cytometry6 cell/ well on a 6-well plate. Then, the concentration of (the best) IC50 was added to each well. After 48 hours, flow cytometry was performed by the BD FACS Calibur Flow Cytometry Machine (BD Biosciences_ USA), and the results were analyzed using the FlowJo.7.6.1 software. We conducted flow cytometry measurements based on the protocols of eBioscience.In order to determine the amount of primary and secondary apoptosis induced under the influence of the extract in each cell line, the cells were grown at a concentration of 1×10RT-PCRΔΔCT method.RT-PCR was performed to identify caspase-3 gene expressions. For this purpose, the cells were collected after 48 hours of treatment with the extract that had the best IC50 on the MTT test, and their total RNA was extracted using the Trizol  method. Then, 1µg total RNA was converted to cDNA using the PrimeScript 1st strand cDNA Synthesis kit  according to kit manufacturer’s instructions. RQ-PCR assay was performed to investigate the level of caspase-3 gene expression in drug-treated and control groups using primers in Table 1. β-2 microglobulin (B2M) was used  as a housekeeping gene for normalization of RT-qPCR data .It should be noted that all tests were performed in triplicate. The fold change Casp3 mRNA in treated cells in comparison with untreated cells was computed by the 2-Statistical analysisAll Experiments were performed in duplicate and repeated three times. IC50 was calculated using the Excel 2013 software, and flow cytometry analysis was done using the FlowJo.7.6.1 flow cytometry software. The data are expressed as mean ± SD for all experiments. GraphPad Prism 5.0  was used to detect significant differences between the control and treated groups. Statistical significance was defined at *P<0.05, **P<0.01, and ***P<0.001 compared to the corresponding controls.Ethyl acetate extract had the best IC50 on the MTT testWhen cells were treated with the aqueous methanol, and ethyl acetate extracts of the Armeniacae semen at concentrations of 0.125, 0.25, 0.5, 1 mg/ml for 48h, the MTT test showed that cell proliferation was inhibited in a dose-dependent manner with all extracts . The besAmygdalin identification by HPLCThe results of the HPLC test indicated the presence of amygdalin in the ethyl acetate extract of Armeniacae semen. There was 0.67% amygdalin in this extract that was calculated as follows: Area sample/ Area STD×C STD.Analysis of apoptosis by double staining KG-1 and NALM-6 cells with annexin V-FITC and PIKG-1 and NALM-6 cells became apoptotic after they were treated with the ethyl acetate extract of Armeniacae semen. The distribution of apoptotic cells measured by flow cytometry showed that the percentage of early (Q3) and late-apoptotic cells (Q2) was 63±1.48% and 32.7±2.31% in KG-1 and 59.22±1.43% and 30.61±1.94% in NALM-6 cell lines, respectively.Effect of Ethyl acetate extract of Armeniacae semen on casp3 gene expressionp<0.05 in KG1 and p<0.001 Nalm6 compared to the untreated control cells.)Molecular examination 48 hours after treatment of KG-1 and NALM-6 cell lines showed a significant increase in caspase-3 gene expression in test samples compared to the controls.  and KG-1 (acute myeloid leukemia) cell lines. 10,11,14,15 . In 2005, Park HJ et al. reported that the extract from the amygdalin has a dose-dependent cytotoxic effect on human colon cancer (SNU-C4)   11 . They also reported that treatment of human chronic myeloid leukemia (K562 cell line) with amygdalin at different concentrations resulted in decrease in cell viability in a dose-dependent manner    14 . In other studies, cytotoxic effect of amygdalin was shown on prostate cell lines (DU145 and LNCaP) and human cervical cancer HeLa cells 10,15.In the present study, based on the evaluation of cell survival by the MTT assay, the cell viability in both KG-1 and NALM-6 cell lines was dose dependent and the survival rate of the cells reduced, with an increase in the concentration of the mixture. Moreover, the best IC50 was related to the ethyl acetate extract in both cell lines. The effect of the extract on the cell viability seems to be dependent on the cell type. Our results are in line with other studies, showing cytotoxic effect of amygdalin on various cancer cell types 7,10,15,16 . Evaluation of the treatment of UMUC-3, TCCSUP, and RT-112 bladder cancer cell lines with amygdalin at concentrations of 1, 10, 25 mg/ml using the Annexin-V-FITC/PI kit showed  a dose-dependent relationship between amygdalin concentration and Annexin V positive cellsSince most anticancer and cytotoxic compounds induce apoptosis in tumor cells, a quantitative study of apoptosis was performed in both KG-1 and NALM-6 cell lines treated with the ethyl acetate extract of Armeniacae semen. Analysis of flow cytometric results in both cell lines indicated an almost 50% apoptosis at IC50. Moreover, investigation of caspase-3 (executive pathway) gene expression at IC50 showed a significant increase in both cell lines compared to controls. The other studies involving other cancerous cell lines also confirmed apoptosis induction and increased caspase-3 activity by amygdalin    10 . Lee et al. showed that amygdalin induces apoptosis in Hs578T breast cancer cells through the caspase-3 pathway    16 . The key role of Caspase-3 in apoptotic pathways is well established. Therefore, the mRNA expression level of Casp3 was assessed to evaluate the effect of the amygdalin extract on the induction of cell death. Our gene expression study showed that the ethyl acetate extract of Armeniacae semen increases the Casp3 at mRNA level. Chang et al. showed that amygdalin increased caspase-3 activity significantly in both DU145 and LNCaP prostate cancer cell lines17,18. Some other studies suggest that rhodanase, which is capable of eliminating cyanide toxicity, exist in normal tissues, but is ineffective in cancer cells. Combined activity of the two enzymes may be responsible for inducing toxic effects in amygdalin-treated cancer cells, while normal cells remain unaffected11,19.The effect of the ethyl acetate extract of Armeniacae semen on PBMCs isolated from normal blood at different concentrations did not show significant cytotoxic effects compared to the negative control. There are hypotheses about the mechanisms of the differences in the response of normal and cancerous cells to amygdalin. Some studies suggest that the cancer cells are rich in β-glucosidase, which is capable of digesting amygdalin to produce cyanide, leading to toxic effects on cancer cells Taken together, the results of the present in vitro study (which never was done before on these types of leukemia) suggest that the ethyl acetate extract of Armeniacae semen have anticancer effects on AML and ALL. However, future studies should focus on the isolation of the effective ingredients of the ethyl acetate extract and evaluation of their effects on the KG-1 and NALM-6 cell lines in human and animal models as well as the assessment of the effect of the extract on the cell cycle. In addition to measuring the expression of caspase-3 gene, the activity of this enzyme should be assessed through the caspase-3 activity measurement kit.


Spatholobus suberectus Dunn are a traditional Chinese medicine activating blood circulation and removing stasis. They have often been used for the traditional Chinese medicinal treatment of breast cancer in modern China. In this study, four novel isoflavanes (1–3 and 5) and four known analogues (4 and 6–8) were isolated from an ethanolic extract of the rattans of S. suberectus. Their structures were elucidated by extensive spectroscopic analyses and electronic circular dichroism studies. MCF-7 and MDA-MB-231 human breast cancer cell lines were used to evaluate the cytotoxic effects of the isolates. Interestingly, compounds 1 and 2 only inhibited the proliferation of MCF-7 cells, while compound 6 showed a selective cytotoxicity against MDA-MB-231 cells. However, compound 4 had significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines.The rattans of  Spatholobus suberectus Dunn (Leguminosae) is a representative traditional Chinese medicine historically used to promote blood circulation and remove stasis. As traditional Chinese medicine theory considers cancer as a kind of disease most possibly related to severe blood stasis, S. suberectus is commonly used for the treatment of cancer in China  colorimetric assay using an estrogen-dependent breast cancer cell line (MCF-7) and an estrogen-independent breast cancer cell line (MDA-MB-231). It is interesting that isoflavanes 1, 2, and 6 showed selective cytotoxicity, and isoflavane 4 inhibited the proliferation of both MCF-7 and MDA-MB-231 cell lines.Isoflavanes and isoflavones are two subclasses of isoflavonoids, which are plant secondary metabolites characterized by a B-ring attached at the C-3 position of the C-ring. They are known for their significant estrogen-like activity, and modern research suggests that they also have antiproliferation in many kinds of human cancer cell lines ,15,16,17es (1–8) , includi1 was isolated as a white amorphous powder, and the presence of hydroxy groups (3446 cm−1) and aromatic rings (1614 and 1495 cm−1) was indicated in its infrared (IR) spectrum. High-resolution electrospray ionization mass spectrometry (HRESIMS) data at m/z 317.1389 [M + H]+  and 339.1208 [M + Na]+  revealed the molecular formula of C18H20O5 for 1. The 1H nuclear magnetic resonance (NMR) spectrum of 1 displayed resonances corresponding to a 1,2,3,4-tetrasubstituted phenyl [δH 6.71  and 6.51 ], a 1,2,4-trisubstituted phenyl , two methylenes , a methine [δH 3.56 (m)], three methoxy groups , and an exchangeable hydroxy group (δH 5.63) (13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 showed 18 carbon signals , 6 × CH (five aromatic methines), and 7 × C (five oxygenated aromatic carbons). These spectroscopic data indicated that compound 1 was an isoflavane possessing a hydroxy and three methoxy groups  and a methylenedioxy  in 2 replaced the 1,2,4-trisubstituted phenyl and the OMe-4′ in 1, respectively. In the HMBC spectrum of 2, correlations of H-3′ with C-1′, C-2′, C-4′, and C-5′; of H-6′ with C-3, C-2′, C-4′, and C-5′; of OMe-2′ with C-2′; and of 4′-OCH2O-5′ with C-4′ and C-5′ confirmed that the methylenedioxy group in 2 was linked to C-4′ and C-5′. The ECD spectrum of 2 exhibited similar Cotton effects  to 1. Although the specific optical rotation -7-hydroxy-8,2′-dimethoxy-4′,5′-methylenedioxyisoflavane.Compound  2 and 1  suggesteguration . Consequ3 was another isoflavane, as indicated by the HRESIMS, IR, 1H-NMR, and 13C-NMR data. Comparison of the 1H and 13C-NMR data of 3 and 1 suggested that the OMe-2′ in 1 was replaced by a hydroxy group in 3. Thus, the planar structure of 3 was established as 7,2′-dihydroxy-8,4′-dimethoxyisoflavane (8-methoxyvestitol), which was reported in 1979   . These sS)-sativan (4) c 0.08, MeOH); CD (MeOH) 238 (Δε +2.05), 284 (Δε −0.98) nm; IR νmax: 3446, 2939, 2839, 1614, 1495, 1462, 1287, 1201, 1173, 1151, 1088, 1036, 966, 928, 824, 803 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 317.1389 [M + H]+ , 339.1208 [M + Na]+ , 355.0949 [M + K]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 1 were shown in (3S)-7-Hydroxy-8,2′-dimethoxy-4′,5′-methylenedioxyisoflavane (2): White amorphous powder, [α]c 0.03, MeOH); CD (MeOH) 242 (Δε +1.86), 283 (Δε −0.62) nm; IR νmax: 3370, 2920, 2851, 1579, 1543, 1467, 1261, 1156, 1116, 1041, 799, 721 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 331.1178 [M + H]+ , 353.1000 [M + Na]+ , 369.0738 [M + K]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 2 were shown in (3S)-7,2′-Dihydroxy-8,4′-dimethoxyisoflavane (3): White amorphous powder, [α]c 0.05, MeOH); CD (MeOH) 234 (Δε +2.43), 284 (Δε −0.50) nm; IR νmax: 3443, 2921, 2851, 1617, 1595, 1463, 1242, 1200, 1165, 1088, 1040, 960, 840, 794, 720 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 325.1052 [M + Na]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 3 were shown in 7,2′-Dihydroxy-8-methoxy-4′,5′-methylenedioxyisoflavan-3-en-2-one (5): Yellow powder, IR νmax: 3257, 2921, 2851, 1711, 1601, 1505, 1463, 1359, 1317, 1239, 1203, 1087, 1033, 975, 857, 803 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 351.0473 [M + Na]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 5 were shown in 50 values represented the mean of three independent replicates.The cytotoxic activity of the isolates against the MCF-7 and MDA-MB-231 human breast cancer cells were determined by a colorimetric MTT assay, as described in the literature ,28. PaclSpatholobus suberectus mainly contains flavonoids, including flavones, flavanones, chalcones, isoflavanes, and isoflavones, some of which have a cytotoxic effect against breast cancer cell lines. In this study, four novel isoflavanes (1–3 and 5) and four known analogues (4 and 6–8) were isolated from the rattans of S. suberectus. Compounds 1, 2, and 6 showed selective cytotoxicity against the estrogen-dependent breast cancer cell line (MCF-7) and the estrogen-independent breast cancer cell line (MDA-MB-231) , while compound 4 had cytotoxicity against both cell lines. Preliminary analysis of the structure–cytotoxicity relationship suggested that C-2′ and C-8 substituents may affect the cytotoxicity and selectivity.


The function of human genes can be strongly inferred from their knockout fitness profiles across hundreds of CRISPR screens, illuminating the modular organization of the cell. Genetic interactions mediate the emergence of phenotype from genotype. The systematic survey of genetic interactions in yeast showed that genes operating in the same biological process have highly correlated genetic interaction profiles, and this observation has been exploited to infer gene function in model organisms. Such assays of digenic perturbations in human cells are also highly informative, but are not scalable, even with CRISPR-mediated methods. As an alternative, we developed an indirect method of deriving functional interactions. We show that genes having correlated knockout fitness profiles across diverse, non-isogenic cell lines are analogous to genes having correlated genetic interaction profiles across isogenic query strains and similarly imply shared biological function. We constructed a network of genes with correlated fitness profiles across 276 high-quality CRISPR knockout screens in cancer cell lines into a “coessentiality network,” with up to 500-fold enrichment for co-functional gene pairs, enabling strong inference of gene function and highlighting the modular organization of the cell. Saccharomyces cerevisiae, systematic genetic deletion studies showed that only ∼1,100 of its ∼6,000 genes (∼20%) were required for growth under laboratory conditions  or between members of parallel pathways (“between pathway interactions”) . When asS. cerevisiae genome has less than one-third the number of protein-coding genes as humans, and despite the quantum leap in technology that the CRISPR/Cas system offers to mammalian forward genetics, yeast remains far simpler to perturb reliably in the laboratory. Several groups have applied digenic perturbation technologies, using both shRNA and CRISPR, to find cancer genotype-specific synthetic lethals for drug targeting . We used the log-likelihood score (LLS) to describe the significance of enrichment. Gene pairs are ranked by Pearson correlation, grouped into bins of 1,000 pairs, and each bin is evaluated for the relative abundance of genes annotated to be in the same KEGG pathway (“true positives”) versus genes annotated to be in different pathways . Data from 13 edges . It has 13 edges . We eval13 edges , 2016.BRAF and related genes that are highly specific to BRAF-mutated melanoma cells , indicating their essentiality in BRAF-mutant cells, supporting efforts to incorporate ERK inhibitors into combinatorial therapies to overcome resistance to targeted BRAF treatments , melanogenesis-associated transcription factor, the developmental Sry-related box (Sox) gene SOX10, and mesenchymal marker ZEB2, indicative of the non-epithelial origin of melanocyte cells and providing insight into the genetic requirements for tissue differentiation in this lineage.The data underlying the Cancer Coessentiality Network is derived from well-characterized cancer cell lines from 30+ lineages, representing the major oncogenic mutation profiles common to those cancers. We expected that many clusters in the network could, therefore, be associated with specific tissues and cancer-relevant genotypes. By testing cluster-level essentiality profiles for tissue specificity , we identified only a small number of clusters that correspond to tissue-specific cancers , which ieatments . This exP < 10−7), contains oncogene FLI1 and tissue-specific TF MYB (P < 10−7) and carries lineage-specific TF PAX8, previously shown to be essential in these cells (P < 10−9), contains β-catenin (CTNNB1) and TF partner TCF7L2   , indicatMYCN, the neuroblastoma-specific paralog of the MYC oncogene , MDM4 , and PPM1D  MYCN, the< 10−44) .TP53 shows the characteristic extreme negative essentiality score of a tumor suppressor gene in wild-type backgrounds, surprisingly, it causes a growth defect when knocked out in three cell lines: HCT1143 breast cancer, PC14 lung cancer, and NB4 AML cells , an oxygen sensor that hydroxylates hypoxia response genes HIF1A and HIF2A, marking them for degradation by the VHL complex in normoxic environments .Although ML cells . All thrdetected . Nor is opy loss . In contkgrounds . The essronments . EGLN1 eKRAS amplifications in pancreatic and colorectal cancer (cluster 276), ERBB2 amplifications in breast and other cancers (cluster 52), and CCNE1 overexpression/RB1 mutation (cluster 101), consistent with well-studied patterns of oncogenesis. All network cluster annotations can be found in the master annotation file (Table S7).These examples indicate the breadth and precision of the coessentiality network but represent results from hypothesis-guided queries. In an effort to learn novel associations from the data, we tested each cluster for its correlation with cell lineage as well as correlation with gene expression, mutation, and copy number amplification of all genes both inside and outside the cluster to identify underlying molecular genetic drivers of modular, emergent essentiality. We identified 270 genes in 30 clusters whose essentiality profiles strongly correlated with their own copy number profiles but not their expression profiles . As copyGiven the underlying data, it is not surprising that oncogenic signatures are clearly evident in the coessentiality network. However, the vast majority of the network structure does not appear to be driven by tissue specificity or mutational signatures. The network contains information complementary to prior functional  and physP-value < 10−6, and fitness profiles have been used to implicate additional members of protein complexes  plus additional subunits of all other ETC complexes. The cluster also contains 49 of 51 subunits of the mitochondrial large ribosomal subunit (P < 10−87), 23 of 25 members of the small subunit (P < 10−39), plus 20 mitochondrion-specific tRNA synthases (P < 10−20). This mitochondrial translation machinery is required for the synthesis of proteins in the ETC complexes. These genes’ inclusion in this cluster, where their essentiality profiles are correlated with those of the complexes they support, reflects a fundamental feature of saturating genetic screens: the essentiality of a given enzyme or biological process is matched by the essentiality of the cellular components required for the biogenesis and maintenance of that process.Epistatic interactions frequently underlie covariation in fitness profiles . ClusterGPX4 shows highly variable essentiality across cell lines , the “21st amino acid,” at its active site. Coessential with GPX4 are all the genes required for conversion of serine-conjugated tRNASer to selenocysteine-conjugated tRNASec , as well as selenocysteine-specific elongation factor EEFSEC, which guides Sec-tRNASec to specific UGA codons , are found in a cluster with 10 PEX genes involved in peroxisome biogenesis, maintenance, and membrane transport , RAB3GAP1 and RAB3GAP2, and guanine nucleotide exchange factor (GEF), TBC1D120 ; canonical mTORC1/mTORC2 regulatory and signaling components PDPK1, AKT1, and PIK3CB; plus G-protein subunit GNB2, previously shown to physically interact with mTOR in response to serum stimulation —is reflected in the anti-correlation of fitness profiles connecting the TSC1/2 cluster and the mTOR cluster.A similar network of modules describes the regulation of the mechanistic target of rapamycin (mTOR), in particular, its detection of cellular amino acid levels . Fig 6 smulation . CanonicMTOR response to cellular amino acid levels is modulated by an alternative pathway that functions at the lysosomal membrane (HOPS complex (VPS26/29/35 retromer complex (RRAGA) and RagC (RRAGC) that transmit information on amino acid abundance to mTORC1 (FLCN) (membrane . We iden complex  and the  complex . This sto mTORC1 . The Rago mTORC1  and foll1 (FLCN) , also me1 (FLCN)  and is, 1 (FLCN)  is refleLCMT1, TIPRL, and PTPA), whose strong connectivity to the TSC1/2 complex may suggest a regulatory role for PP2A in MTOR signaling. PP2A has previously been posited to be an activator of TSC1/2 upstream of MTOR . IGF1R is a highly N-glycosylated RTK, and the IGF1R complex is tightly connected to the OST complex in our network. EGFR, also highly glycosylated . Prior work suggests that membrane glycolipid composition can strongly influence EGFR autophosphorylation and signaling . Although the coessentiality network does not capture a large portion of protein–protein interactions  or genetReconstructing biological processes from coessentiality information has some limitations. First of all, interactions between genes that do not affect cell fitness cannot be captured. Second, interactions between gene pairs that are not perturbed in the cell line pool cannot be captured. Last, the presence of genetic alterations like mutation or copy number amplification can generate confounding effects. A critical next step will be to understand the underlying context that drives the emergent essentiality of specific bioprocesses in specific backgrounds. The health implications of this question are profound. In cancer, to understand the causal basis of modular emergent essentiality is to identify matched pairs of biomarkers  and precision targets  for personalized chemotherapeutic treatment. In addition, lineage-specific essential processes could provide explanatory power for germline mutations causing tissue-specific disease presentation, in cancer as well as other diseases.https://hartlab.shinyapps.io/pickles/ (Expanding the coverage of the network will require different screening approaches. Fitness screens in cancer cell lines in rich media will miss cellular dependencies that are present only under stress conditions. In yeast  and nemapickles/  and downhttps://figshare.com/articles/_/5520160). We only kept protein-coding genes for further analysis and updated their names using HGNC (https://github.com/hart-lab/bagel). In comparison with published BAGEL version v0.92  (Table S5). All network figures shown in this article were drawn using Cytoscape , which is the harmonic mean of precision and recall at Bayes factor 5. Then, among 338 cell lines, 276 cell lines were selected for further study, by F-measure (>0.85) and the number of essential genes , to prevent noise from marginal quality of screens .Table S4 Network interactions of coessentiality network.Table S5 Performance benchmark of coessentiality network was conducted by a pathway enrichment test using the KEGG pathway database (downloaded in 2015). Pathways having >200 annotated genes  were discarded to minimize bias. For each network, gene pairs were ranked by PCC and binned into groups of 1,000 pairs. The cumulative LLS  was calcPreprint; https://portals.broadinstitute.org/achilles). For Wang et al screens, we downloaded raw read counts from their article. For https://depmap.org/portal/). We calculated Bayes factor profiles from raw read counts of screens and controls defined in articles through the BAGEL pipeline. To filter low-quality screens out, we applied thresholds of F-measure (BF = 5) 0.80 for To compare performance with other screens, we constructed coessentiality networks for two other CRISPR data sets  and threNetwork clustering was conducted with the Markov Cluster Algorithm (MCL) . We firsGene clusters derived by MCL algorithm.Table S6 Pathway annotations  of major clusters.Table S7 P-values of differential expression, copy number, and mutation between mean essentiality of clusters and gene properties. For RNA-seq expression, we used log2(FPKM + 0.5). For copy number variation, we discretized copy number value into three classes . For mutation data, we made a binary profile of mutation presence, treating silent mutations as wildtype. To measure significance of associated factors, we conducted a t test for expression data, Fisher’s exact test for copy number , and Fisher’s exact test for binary mutation data.To identify molecular genetic factors associated with cluster essentiality, we downloaded RNA -seq , copy number variation, and mutation profiles from the Cancer Cell Line Encyclopedia (CCLE) database  in 2017.P-value was measured by Bonferroni correction of P-value.Tissue-specific essentiality was calculated for 517 clusters and 3,483 essential genes against 19 tissues: upper aerodigestive tract, thyroid, large intestine, autonomic ganglia, soft tissue, central nervous system, haematopoietic and lymphoid tissue, stomach, endometrium, liver, urinary tract, bone, lung, breast, skin, oesophagus, ovary, kidney, and pancreas. For each cluster, we first calculated mean essentiality of member genes per cell line. Then, for each tissue, we conducted Wilcoxon rank sum tests of two groups, a group belonging to the target tissue type, and a group consisting of all other tissue types. Last, adjusted To investigate how many interactions are overlapped with the coessentiality network from Avana screens, we downloaded functional interaction networks  were kept for further steps. To prevent abnormal outlier or zero values, we gave 0.5 pseudo count to all genes, divided each value by the mean of each gene, and took the log form of the resulting value. Expression similarity for all possible combinations of two genes was measured by PCC . Proteinhttps://www.cancerrxgene.org/gdsc1000/GDSC1000_WebResources/Home.html.Drug log(IC50) values used for correlation analyses were taken from the Genomics of Drug Sensitivity in Cancer (GDSC) database . Data tastats (version 3.2.3), based on mean gene BF in a cluster in a cell line against the matching cell line log IC50 value of each drug. Correlations calculated had between 34 and 187 data point pairs  within the overlapping 192 cell lines between GDSC database and Avana project. Pearson correlations that resulted in a negative correlation with a P-value less than 10−4 were added to the annotation text file. Negative correlations imply that IC50 values decrease as mean cluster BF in a given cell line increases . Correlations were calculated for the top 309 co-correlation ranked clusters.Cell line annotation style from TableS4A was altered to match Avana project cell line annotation style. GDSC data contained log (IC50) values from 990 cell lines, which overlapped with 192/276 cell lines used in the Avana project. Log IC50 values contained 265 unique GDSC drug IDs with 250 unique drug names. Pearson correlations were computed using cor.test from the R package https://portals.broadinstitute.org/ccle/data. We used the gene-centric RMA-normalized expression data. Also, we used preprocessed RNA-seq and drug information (log IC50 data) used for other analysis in this article.The CCLE Reverse Phase Protein Array (RPPA) data, RPPA antibody information, and cell line annotations of 1,037 cancer cell lines were retrieved from the CCLE portal at: Essentiality scores for each gene were preprocessed using quantile normalization within each cell line. The quantile-normalized essentiality scores for the selected genes for each of the 276 cell lines were gathered in a matrix. Cell lines with essentiality scores lower than or equal to −10 were set at a Bayes factor of −10.Next, the heat map was plotted sorting the cell lines by the mean Bayes factors for each gene in the cluster by using the matplotlib package in Python. The heat map was annotated by the presence of TP53 mutations (orange) with missing values as black and the Nutlin 3a natural log half maximal inhibitory concentration (IC50) values for each cell line with missing values as light grey.MYC gene was added to Cluster 43 (MYCN) genes, and their quantile-normalized essentiality scores were gathered in a matrix for all high-quality Avana project cell lines. The heat map was plotted sorting the cell lines by the mean Bayes factors for each gene in the cluster. The heat map was annotated with MYC and MYCN expression values as well as a tissue key, specifying the neuroblastoma cell lines in orange.From the 64k network, the From the 64k network, the quantile-normalized essentiality scores of genes from Cluster 17 were gathered in a matrix for all high-quality Avana project screens. The heat map was plotted sorting the cell lines by the mean Bayes factors for each gene in the cluster and annotated by a tissue key specifying the cell lines from the hematopoietic and lymphoid tissues in orange.ERBB2gene, the RPPA values for ERBB2, expression values for ESR1 and CP724714, and Refametinib log IC50 values for each cell line with missing values as light grey.Clusters 52 and 55 from the 64k network were combined and the quantile-normalized essentiality scores for each gene in the clusters were gathered in a matrix after filtering for the cell lines from the breast tissue. The heat map was plotted by sorting with the mean essentiality scores for the genes in the clusters across the breast cell lines. The heat map was annotated with the log2 copy number values for the BRAF, and the log IC50 values for PLX-4720, with missing values in light grey.From the 32k network, quantile-normalized essentiality scores for the genes in cluster 19 were gathered in a matrix for all high-quality Avana cell lines. The cell lines were sorted by the mean Bayes factors and a heat map was plotted. The heat map was annotated with log2 copy number, RPPA values, presence of mutation (in orange) for We filtered for the cell lines in which at least one of the following RTKs: EGFR, ERBB2, ERBB3, FGFR1, IRS2, and IGF1R have a BF of greater than 20. Then for that subset of cell lines, the essentiality scores of the RTKs and their downstream effectors were gathered in a matrix. Next, in Python, the hierarchical clustering package called scipy.cluster.hierarchy was used to cluster the cell lines for each RTK. We used the “average” method representing the UPGMA algorithm and the “Euclidean” distance metric to calculate the distance between the newly formed cluster and each remaining cluster and perform hierarchical clustering. The clustered heat maps were annotated for EGFR and ERBB2 copy number data.We filtered for the cell lines in which at least one of the following genes: KRAS, NRAS, BRAF, and PIK3CA had a BF of greater than 20. Next, we obtained the essentiality scores of the RTKs and their downstream effectors for the filtered cell lines. Later, we performed hierarchical clustering to cluster the cell lines for each RTK as explained above. Next, the clustered heat maps were annotated with mutation status for KRAS, NRAS, BRAF, and PIK3CA in orange.P-values were calculated using the scipy.stats.pearsonr from the scipy package based on the mean cluster essentiality score in a cell line against F-measure value of the matching cell line.For each cluster in the network, the mean Bayes factor of the genes in that cluster was calculated to get a mean essentiality score for the cluster for each cell line. Then, the Pearson correlations and corresponding


Under normal conditions our intestines are inhabited by trillions of diverse microorganisms composing the intestinal microbiota, which are mostly non-pathogenic anaerobic commensal bacteria vital for the maintenance of immune homeostasis. The composition and diversity of the intestinal microbiota can be disturbed by various factors including diet, antibiotics, and exposure to intestinal pathogens. Alterations of the intestinal microbiota contributes to many diseases including graft-vs.-host disease (GVHD), a life threatening complication that occurs after allogeneic hematopoietic cell transplantation  caused by an allogeneic reaction of donor T cells against recipient target tissues. Intestinal GVHD is most difficult to treat and connected to a high mortality. Due to recent advances in high-throughput sequencing technology, composition of the microbiome during allo-HCT has been characterized, and some common patterns have been identified. Metabolites produced by intestinal bacteria were shown to promote intestinal tissue homeostasis and immune tolerance post-allo-HCT. In this review, we discuss the role of the intestinal microbiota and metabolites in the context of acute GVHD. Moreover, novel therapeutic approaches that aim at protecting or regenerating intestinal cell populations will be highlighted. Allogeneic hematopoietic cell transplantation  is the only curative therapy option for most acute leukemias. Prior allo-HCT patients undergo a conditioning regimen, consisting of chemotherapy with or without radiotherapy to decrease the number of malignant cells, and prevent rejection of the transplanted hematopoietic donor cells. Subsequently, hematopoietic stem cells (HSCs) from the bone marrow or peripheral blood of an allogeneic donor are infused. In addition to the HSCs, the graft contains allogeneic donor T cells, which can on the one hand attack residual malignant cells, known as graft-vs.-leukemia (GvL) effect, but on the other hand may attack healthy host tissues, resulting in graft-vs.-host disease (GVHD). Although the understanding of the disease pathophysiology has improved significantly in the last decades, acute GVHD is still a major cause of non-relapse morbidity, and mortality post-allo-HCT . In partThe important influence of intestinal microbiota on immune responses, including post-allo-HCT, becomes more and more recognized and the gut is one of the main targets of acute GVHD. Severe gastrointestinal (GI) GVHD remains a major issue after allo-HCT, since it is difficult to treat and involvement of the GI tract is reported in almost all fatal acute GVHD cases . In addiThe first hint that the intestinal microbiota affect GVHD development dates back to the early 1970s, when first studies in murine models showed that GVHD was reduced and survival was prolonged in antibiotics-treated or germ-free mice undergoing allo-HCT –13.Consequently, efforts were made to translate these insights into the human setting. Clinical trials investigated gut decontamination using antibiotics or laminar-airflow isolation rooms for patients undergoing allo-HCT, in order to minimize contact to microbiota –19. HoweHowever, interest in the interplay between the microbiome and inflammatory conditions has been growing again in recent years. Major advances in modern high-throughput sequencing technology made it possible to investigate changes in the composition of the intestinal microflora after allo-HCT in depth and to analyze which bacteria might be detrimental and which may be beneficial with respect to immune tolerance post-allo-HCT.Firmicutes and Bacteroidetes phyla  preceding the development of GVHD in pediatric allo-HCT patients , also known as stool transplantation, has been used to successfully treat recurrent patients . Very repatients , 45. A prelieved . AnotherMicrobial metabolites comprise a multitude of different intermediate products and end products of intestinal microbiota metabolism with various functions, yet their role in GVHD pathophysiology is merely starting to be discovered. Very recent publications revealed that one of the mechanisms, how intestinal bacteria can influence immune tolerance post-allo-HCT, is the production of metabolites Figure . Table 2Il22 transcription and mucosal immune homeostasis  . Daily iFocusing more on the receptors of bacterial metabolites, the group of Dr. Reddy recently revealed an important regulatory role for GPR43, a G-protein-coupled receptor on IECs recognizing the microbiota-derived short-chain fatty acids, butyrate, and propionate, during GVHD development. Sensing of the microbial metabolites by GPR43 reduced GVHD severity and mortality in murine models by activating the NLRP3 inflammasome in recipient non-hematopoietic cells via ERK phosphorylation. Importantly, GPR43 did not seem to play a role for host and donor hematopoietic cells, since absence of GPR43 did not lead to systemic activation of donor T cells or inflammation . While pIn a recently published study, Varelias et al. demonstrated that recipient mucosal-associated invariant T (MAIT) cells are present in acute GVHD target organs in murine models, including the intestinal lamina propria . These cSevere GI GVHD remains a major issue after allo-HCT, since it is difficult to treat and involvement of the GI tract is reported in almost all fatal acute GVHD cases . TherefoIntestinal stem cells (ISCs) represent important players for both physiological renewal of intestinal cells and tissue regeneration after injury. In a murine model, it was shown that ISCs are damaged during the conditioning regimen and the development of GVHD . This coIL-22 is a member of the IL-10 cytokine family that plays opposing roles in different autoimmune diseases, being able to drive or regulate disease –73. Simi+ ISCs (in vivo treatment approaches and showed the feasibility of both recombinant IL-22 and a recombinant human IL-22-dimer/Fc-fusion protein (F-652) for the treatment of murine GVHD. Based on the results from these preclinical studies, a Phase IIa clinical trial investigating F-652 in combination with systemic corticosteroids in patients with grade II-IV lower GI acute GVHD is currently recruiting (NCT0240665).In contrast, Hanash et al. demonstrated that recipient innate lymphoid cell (ILC)-derived IL-22 was able to enhance regeneration of IL-22R-expressing ISCs in a more acute major mismatch GVHD model . Host de+ ISCs . MoreoveThe described conflicting data of both a regulatory and inflammatory role for IL-22 in GVHD might result from many factors differing between the studies, including kinetics of the model, source of IL-22, presence or absence of other cytokines and pathophysiological differences between acute and chronic GVHD models.Reg3γ deficient mice this protection was completely abrogated, emphasizing the important role of REG3γ for gastrointestinal crypt homeostasis  to regulate GI acute GVHD . REG3α beostasis .REG3A SNPs were not associated with acute GVHD severity in this retrospective study including 350 patients  in the genes encoding two of the most abundant Paneth cell antimicrobial peptides REG3α and HD5 were studied . Interespatients .Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family, regulating differentiation, and proliferation of epithelial cells, including IECs, as well as intestinal stem cells , 84. AlmIn the last 10 years it has become evident that a loss of diversity of the intestinal microbiota flora due to reduced food intake, chemotherapy-related damage, and antibiotics promotes the development of GVHD, which changed the dogma that antibiotics-based decontamination of the intestinal tract improves outcome post-allo-HCT. With the discovery that indoles and butyrate promote intestinal homeostasis the connection between antibiotics treatment and unfavorable outcome post-allo-HCT could be explained. Beyond bacterial species that were connected to GVHD, recent studies have also identified fungi and viruses that occurred more frequently in patients with severe GVHD. Based on this improved understanding on how the microbiome and the intestinal tract interact, novel strategies have been developed such as FMT that hold promise to overcome acute GVHD in a majority of patients. Ongoing clinical studies will provide important information on the role of FMT and regenerative strategies.NK and RZ collected literature, discussed the studies, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation.CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb.  CCNE1, and reduced expression of another, called Rb. The ratio of CCNE1:Rb expression proved to be an accurate prognostic factor among patients enrolled in a clinical trial of palbociclib, performing better than either CCNE1 or Rb levels on their own.The expression levels of two genes involved in controlling the cell cycle offers a promising predictive indicator of whether women with hormone receptor–positive breast cancer are likely to develop resistance to the drug palbociclib. A team led by Luca Malorni and Ilenia Migliaccio from the Hospital of Prato, Italy, analyzed gene and protein profiles in a panel of breast cancer cell lines with differential sensitivity to palbociclib. The researchers found that, compared to palbociclib-sensitive cell lines, those that were drug-resistant tended to have elevated expression of one cell-cycle–related gene, called  However, acquired resistance to these agents is universal, and results from clinical trials indicate that approximately 10 to 15% of patients may exhibit de novo resistance.4Palbociclib is a highly specific, orally active CDK4/6 inhibitor currently approved for the treatment of hormone receptor-positive, HER2-negative (HR+/HER2neg) advanced breast cancer (BC) in combination with the endocrine agents letrozole or fulvestrant.5 The primary target of CDK4/6 action is the retinoblastoma susceptibility gene product (Rb) and other Rb family members (such as p107 and p130).6 Phosphorylation of Rb (pRb) by active cyclin-CDK complexes leads to release of transcription factors of the E2F family, and transcription of genes required for S-phase entry.6 Constraint upon CDK activity and G1 progression is provided by the universal CDK inhibitors of the Cip-Kip family, including p21 and p27, and the specific CDK4/6 inhibitors of the Ink family typified by p16.7CDK4 and CDK6 are kinases activated by binding to D-type cyclins, bearing a crucial role in cell proliferation through the regulation of cell cycle entry.10 However, these events have been observed only in a limited number of in vitro and in vivo models, and these data have not been fully validated in the clinical setting. Therefore, the aim of our study is to further investigate if common cell cycle alterations, especially in the Rb/E2F pathway, could be observed in palbociclib-resistant models using a large panel of HR+ BC cell lines.Preclinical data have already shown that deregulation of cell cycle genes and proteins is associated with resistance to palbociclib, including overexpression of cyclin E1 and loss of Rb.We challenged eight BC cell lines with palbociclib including T47D, ZR75-1, MCF7, MCF7 Estrogen Deprivation Resistant (MCF7 EDR), MCF7 Tamoxifen Resistant (MCF7 TamR), CAMA1, MDA MB 361, and BT474. We developed PDR derivatives for all BC cell lines with the exception of CAMA1, which failed to restore growth with 350 nM palbociclib over two separate attempts. The time to develop resistance for all other models ranged from 10 to 27 weeks.P-value < 0.05), while PDR derivatives did not show significant differences in cell growth when treated with palbociclib 1 µM compared to vehicle-treated cells  lower proportions of β-galactosidase positive cells during palbociclib treatment compared to their PDS counterparts, demonstrating the ability to revert the palbociclib-induced senescence-like phenotype  or with palbociclib starting treatment concentration (STC) for 3 days (PDS treated) and from PDR cells continuously receiving palbociclib 1 µM.CCNE1 and RB1 were consistently deregulated in all PDR models versus PDS treated cell lines  ; similarly, genes down-regulated at the time of resistance were also significantly enriched in E2F targets indicating that the pathway remains inhibited by palbociclib at the time of resistance  Fig. . Interes05) Fig. , while t05) Fig. .CCNE1 expression being a common event.Overall these data suggest that palbociclib effectively inhibits cell growth of HR+ BC cells through inhibition of the Rb/E2F pathway. However, at the time of resistance, signaling is only partially restored, with increased To validate the role of cell cycle-related proteins during palbociclib treatment and at the time of resistance, we performed western blot (WB) analysis for selected markers in PDS cell lines treated with palbociclib for 3 and 6 days at the STCs, and in PDR models continuously receiving palbociclib 1 μM.14 were consistently downregulated by palbociclib. Conversely, cyclin D1 and cyclin E1 levels tended to increase during treatment. All other markers were modulated heterogeneously, or were not modulated by the treatment , these markers significantly correlated with higher IC50 values for palbociclib  and among all drugs tested in the dataset20 palbociclib showed the strongest association with CCNE1/RB1 ratio . In addition, analyzing GE data from BC cell lines of the Iorio et al. dataset,20 but taking into account the IC50 values for palbociclib obtained from a previous BC-focused study,19 we were able to demonstrate that CCNE1, RB1, and CCNE1 /RB1 ratio correlated with higher IC50 also in BC cell lines  was found in patients with a higher CCNE1/RB1 ratio  showing a poorer survival compared to those with a ratio <1 , before starting palbociclib (C1D1) and after 2 weeks of palbociclib (C1D15), and surgical samples were analyzed for Ki67, GE, and mutation profiles.23 We found that CCNE1/RB1 was significantly higher in patients resistant to palbociclib at C0D1, C1D1, C1D15 (p(WMW) = 0.0048; p = 0.013; p = 0.032, respectively, Fig. CCNE1/RB1 performed better than CCNE1 and RB1 alone in discriminating palbociclib-resistant versus sensitive patients at C0D1 (AUC(CCNE1/RB1) = 0.92; AUC(CCNE1) = 0.83; AUC(RB1) = 0.69), and at C1D1 (AUC(CCNE1/RB1) = 0.92; AUC(CCNE1) = 0.75; AUC(RB1) = 0.83), while showing equivalent performance compared to CCNE1 at C1D15 (AUC(CCNE1/RB1) = 0.9; AUC(CCNE1) = 0.9; AUC(RB1) = 0.55)  Fig. .26 We obtained reliable models of acquired resistance to palbociclib, as revealed by functional analyses investigating proliferation, IC50 and β-galactosidase expression, for all cell lines except CAMA1. Yang et al. succeeded in the development of models of CAMA1 resistant to abemaciclib,10 probably due to the different approach used10 and the different inhibitory profile of abemaciclib.27In this study, we developed a panel of HR+ BC cell lines with acquired resistance to palbociclib by chronically exposing cells to increasing doses of the drug. To account for the heterogeneity of luminal BC, we selected eight BC cell lines with different initial degrees of sensitivity to palbociclib. We deliberately challenged cell lines to palbociclib alone, rather than combined long-term estrogen deprivation (LTED) and palbociclib in order to identify markers specifically related to palbociclib resistance, thus avoiding the confounding effect of endocrine treatment. We acknowledge that the resultant models might not fully recapitulate the clinical scenario wherein CDK4/6 inhibitor resistance occurs in the setting of dual hormonal therapy and palbociclib. However, we did include two models previously resistant to hormonal therapy in order to account for the effect of combined palbociclib and endocrine resistance. Two HR+/HER2+ cell lines were also included to search for strong common mechanisms of resistance that might occur in HR+ BC despite HER2 status, given that palbociclib has also shown efficacy in HER2+ BC models.29 As expected, and in line with previous reports,31 we observed an inhibition of this pathway in response to palbociclib. We observed a marked reduction of the RBsig, a gene-signature of loss of RB1 function12 and found an enrichment in E2F targets within genes downregulated by palbociclib, consistent with previous observations.30 The activity of palbociclib on the Rb/E2F pathway was confirmed by WB, as revealed by decreased levels of total Rb coupled with decreased phosphorylation of Rb on S807/811 after 3 and 6 days of treatment. Together with Rb, we also observed a general decrease in p107 levels in all cell lines. As already reported by previous studies with cell lines sensitive to CDK4/6 inhibitors,31 our PDS models displayed cyclin D1 accumulation, reduction of E2F-regulated proteins (such as cyclins A1 and E2 and CDK2), and p130 stabilization when receiving palbociclib.As a first step, we explored the effects of palbociclib on the Rb/E2F pathway, which is the target of CDK4/6 inhibitors.9 Our PDS models showed a persistent inhibition of the Rb/E2F pathway after 3 and 6 days of treatment. However, we did not analyze earlier time points, and therefore cannot exclude a partial recovery of the pathway at 72 h as suggested by Herrera-Abreu et al.9It has been demonstrated that BC cells show adaptation following CDK4/6 inhibition as early as 72 h. This has been shown to be mediated by non-canonical activation of cyclin D1/CDK2 complexes, which in turn induce a recovery of Rb phosphorylation and S-phase entry despite CDK4/6 inhibition.33 In particular, cyclin E overexpression and Rb loss have been previously indicated as possible mechanisms of acquired resistance to CDK4/6 inhibitors in some BC cell lines.10 Our GE and WB data showed that both cyclin E1 overexpression and Rb underexpression occur across different and heterogeneous BC cellular models, including HER2+ cells, which has not been shown previously. We also demonstrated that CCNE1 amplification and RB1 deletion might explain only partially these findings, suggesting that other mechanisms could be implicated. Intriguingly, these genomic alterations occurred in both HER2+ and HER2neg cell lines, again supporting the hypothesis that strong mechanisms of acquired resistance to palbociclib might occur in HR+ BC independent of HER2 status. This is of potential clinical relevance as CDK4/6 inhibitors are currently under investigation for patients with HR+ /HER2+ BC.34It has already been shown that alterations in cell cycle genes and proteins are associated with acquired resistance to palbociclib.32At the time of resistance, we observed a slight reactivation of the Rb/E2F pathway as demonstrated by the increased RBsig expression in PDR cells, and by the modulation of some of the E2F targets analyzed. However, the pathway remained significantly inhibited compared to the PDS untreated cells, indicating that palbociclib treatment may still be inhibiting the pathway. This is consistent with previously published data in ovarian cancer, wherein cells resistant to palbociclib did not fully restore E2F target gene expression.31 Intriguingly, phosphorylation status of p27 has also been suggested as a mechanism of resistance to CDK4/6 inhibitors.35Another common marker that we found at the time of acquired resistance is the reduced expression of p27. In a previous study, Dean et al. similarly suggested that loss of CDK inhibitors p21 and p27 may foster the ability of cells to escape from palbociclib-induced cell cycle arrest.36 Loss of RB1 has been already suggested as a mechanism of de novo resistance to these drugs.19 Here, we identified CCNE1 as an additional potential marker of de novo resistance in a large panel of pan-cancer and BC cell lines. To date, the role of cyclin E1 as a marker of de novo resistance has been demonstrated in other tumor types such as ovarian and gastric cancer38 but only suggested in BC.39 Our data suggest that both CCNE1 and the CCNE1/RB1 ratio, which evaluates both CCNE1 and RB1, might serve as “pan-cancer” markers of de novo resistance to palbociclib and that the CCNE1/RB1 ratio might serve as better marker compared to the single biomarkers CCNE1 and RB1 used separately, both in BC and in other cancer types. In BC, this is evident for palbociclib IC50 values from Finn et al,19 but not when values from Iorio et al.20 were considered  upon which the patient had experienced previous progression as a prior line of therapy for ER+/HER2neg metastatic BC. This might be a potential additional setting to validate the role of the CCNE1/RB1 as a marker of de novo resistance to palbociclib.One limitation of our study is the lack of clinical validation of our findings on the role of 41 and to palbociclib in vitro.42 Similarly, overexpression of LMW cyclin E detected by immunohistochemistry has been associated with a lesser benefit from treatment with palbociclib and endocrine therapy in patients with metastatic BC.42 In our WB studies of the PDR models, LMW isoforms of cyclin E1 were not detected, suggesting that the full length is predominantly overexpressed in our models of acquired resistance. However, our in silico analyses could not exclude the possibility that LMW cyclin E may contribute to the effect on de novo resistance we described for cyclin E1, as both RNA and RPPA data cannot distinguish between the full length and the LMW isoforms.Recent data have shown that expression of the cytoplasmic low-molecular-weight isoforms (LMW) of cyclin E is associated with resistance to aromatase inhibitorsCCNE1/RB1 ratio have poorer OS compared to patients with a ratio <1, suggesting that this may represent a population of patients with an adverse outcome, which might mandate tailored therapeutic strategies. However, more detailed studies are necessary to dissect the prognostic versus predictive value of this biomarker in early BC, which would be of particular interest since CDK4/6 inhibitors are currently being tested in the adjuvant setting.43We demonstrated that patients with early ER+ BC with a high CCNE1 overexpression/amplification and reduced expression/loss of RB1 as common events that occur at the time of acquired resistance in luminal BC cell lines, independent of HER2 status. Furthermore, we have identified the CCNE1/RB1 ratio as a potential marker of de novo resistance to palbociclib both in vitro and in clinical samples.The strengths of our study include the size of the employed cell lines panel: to the best of our knowledge, this is the largest panel of HR+ BC cell lines ever developed to investigate acquired resistance to palbociclib. In turn, we have expanded previous knowledge on acquired resistance to palbociclib by confirming Results of our study, if further confirmed and tested in clinical trials, might lead to the identification of new biomarkers of de novo and acquired resistance to palbociclib, which would assist to better personalize treatment of patients with HR+ BC.44 MDA MB 361 were purchased from Sigma-Aldrich and BT474 from Interlab Cell Line Collection, Genova, Italy.T47D and ZR75-1 cell lines were obtained from Dr Livia Malorni, CNR Avellino, Italy; CAMA1 were obtained by American Type Culture Collection; MCF7 parental cells, MCF7 EDR and MCF7 TamR were previously described.Cells were cultured at 37 °C and 5% CO2. T47D, ZR75-1, MCF7, CAMA1, MDA MB 361, and BT474 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/glucose and L-glutamine (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone) and, 10,000 U penicillin and 10 mg steptomycin/mL solution (P/S) (Sigma-Aldrich). MCF7 EDR and MCF7 TamR were grown in DMEM with 4,5 g/glucose and without L-glutamine and phenol red (Lonza) supplemented with 10% charcoal-stripped FBS (CS-FBS) (GIBCO), and P/S. MCF7 TamR cell medium was supplemented with 100 nM final concentration of (Z)−4-Hydroxytamoxifen (Sigma-Aldrich) dissolved in 100% ethanol.PDR were generated by long term culturing of cells with increasing concentrations of palbociclib from the STC up to 1 μM. STC was preliminary assessed on each model and defined as the concentration that, after 9 days of treatment, inhibited cells growth rate between 40 and 60%. Cells were considered resistant to palbociclib when they restored the ability to regularly grow in the presence of palbociclib 1 μM.Palbociclib STC was 50 nM for T47D, ZR75-1, MCF7 EDR, and MCF7 TamR cell lines, 100 nM for CAMA1 and 350 nM for MCF7, MDA MB 361 and BT474 cell lines. PDR cells were then continuously cultured in their original media with the addition of palbociclib 1 μM. Palbociclib was provided by Pfizer and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich).In January 2016, all cell lines and their PDR derivatives have been authenticated by short tandem repeat DNA profiling analysis. This analysis was performed in service by BMR Genomics, Padova, Italy.For proliferation and IC50 assays a total of 3000 cells/well of PDS or PDR cell lines, cultured in their individual media, were plated in 96-well plates in triplicate, 24 h before beginning treatments. These consisted of palbociclib 1 µM, palbociclib STC or 0.01% DMS0 (vehicle) for 6 days for proliferation assays or doubling dilutions of palbociclib, from 64 µM to 0.97 nM for 9 days for the IC50 assays. Media were replaced every 72 h. Cells were fixed with 4% glutaraldehyde grade II (Sigma-Aldrich) and stained with 0.05% methylene blue (Sigma-Aldrich). The dye was subsequently extracted with 3% HCl (Carlo Erba) and absorbance measured at 655 nm. Three biological replicates for each PDS or PDR cell line were performed.PDS and PDR cell lines were plated in 12-well plates in duplicate, in a range of 10,000–20,000 cells/well. Cells were plated in their individual medium which was replaced after 24 h with medium containing palbociclib 1 µM or 0.01% DMSO as a control. Beta-galactosidase activity was assessed at day 6 by Senescence detection kit (BioVision) according to the manufacturer’s instructions. Positive and negative cells were counted in six randomly selected 20X fields and the percentage of positive cells was calculated. Three biological replicates for each PDS or PDR line were performed.TM MiniPrep kit (Zymo Research), according to the manufacturer’s instructions. DNA concentrations and quality were checked by spectrophotometer.DNA was isolated from PDS and PDR cells grown in their individual media supplemented with 0.01% DMSO or palbociclib 1 µM, respectively. DNA extraction was performed with the Quick-gDNATM RNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions. RNA quantification and quality analysis were performed on a Bioanalyzer 2100 System (Agilent), using the RNA 6000 nano Kit (Agilent). cDNA synthesis and biotin-labeled cRNA generation were performed using the Illumina TotalPrep RNA Amplification Kit (Ambion), according to the manufacturer’s protocol using 500 ng of total RNA. Quality assessment and quantification of cRNAs were performed with Agilent RNA kits on the Bioanalyzer 2100 System. Hybridization of cRNAs (750 ng) was carried out using Illumina Human 48k gene chips (Human HT-12 v4 BeadChip). Array washing was performed using Illumina High Temp Wash Buffer for 10′ at 55 °C, followed by staining using streptavidin-Cy3 dyes (Amersham Biosciences). Probe intensity data were obtained using the Illumina GenomeStudio software.RNA was isolated from PDS cells treated with 0.01% DMSO, as a control, or with palbociclib at the STC for 3 days, and from PDR cells at early (before or at passage 15) or late (after passage 15) stage of resistance, grown in their individual media supplemented with palbociclib 1 µM. The number of passages of PDR cells was calculated starting from the moment they restored the ability to regularly proliferate in the presence of palbociclib 1 µM. RNA was extracted from cells with the Direct-zolP-value < 0.05 in at least one sample were considered for downstream analysis (n = 31016). In case of multiple probes matching to a single gene, the probe with the highest signal-to-noise ratio was taken into consideration. To mitigate the effect of cell line specific expression profiles, the log2-ratio values of normalized expression of treated PDS and PDR to the corresponding untreated PDS GE profiles were computed. . The list KEGG_CELL_CYCLE (http://software.broadinstitute.org/gsea/msigdb/cards/KEGG_CELL_CYCLE) was used to identify cell cycle pathway genes. Top variable cell cycle genes  was performed by cubic spline algorithm implemented in the Illumina GenomeStudio software version 1.9.0. Only probes showing a detection nes Fig.  were defhttp://software.broadinstitute.org/gsea/msigdb/cards/HALLMARK_E2F_TARGETS) as gene set. Differential expression between log2-ratio expression values was tested by Wilcoxon–Mann–Whitney (WMW) test.E2F gene set enrichment was computed with Gene Set Enrichment Analysis (GSEA) version 2-2.2.3 by using log2-ratio values of averaged (mean) FC across PDS treated and PDR cell line models as ranking parameters and HALLMARK_E2F_TARGETS . A volume of 15 μg of proteins were separated under denaturing conditions by electrophoresis on 10% or 7.5% polyacrylamide gels  and transferred to nitrocellulose membranes  by electroblotting . The blots were checked using the ChemiDoc MP Imaging System and the Image Lab software (Bio-Rad) to confirm uniform transfer of all samples. Blots were then blocked with Tris-buffered saline (Sigma-Aldrich) containing 0.1% Tween 20 solution (Sigma-Aldrich) and 5% non-fat dry milk  or 5% BSA (Santa Cruz Biotechnology) for 1 h at room temperature (RT) and then incubated with primary and secondary antibodies.PDS cells treated with 0.01% DMSO, as a control, or with the STC of palbociclib for 3 or 6 days and PDR cells grown in their individual media supplemented with palbociclib 1 µM were lysed with RIPA buffer , supplemented with 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and 1% HaltTM software v 5.2.1 (Bio-rad). WB were repeated in two biological replicates. A list of primary and secondary antibodies with their working conditions is provided in Supplementary Table Analysis of WB bands and normalization to total proteins were performed using the Image LabRB1 and CCNE1 between PDR and PDS cells were assessed with RB1, CCNE1 and the reference gene EIF2C1 CNV Assays on a QX200TM droplet digital PCR (ddPCR) system (Bio-Rad). PCR reactions were prepared with approximately 10 ng of DNA and ddPCR supermix for probes (Bio-Rad). RB1 and CCNE1 probes (Bio-Rad) were stained in FAM while EIF2C1 probe (Bio-Rad) was stained in HEX. The ratios CCNE1/EIF2C1and RB1/EIF2C1 were calculated using Quantasoft software (Bio-Rad). At least one negative control with no DNA was included in each run. Experiments were performed three times in duplicate.Copy number alterations of P-values < 0.05 were considered significant.For proliferation assays, growth fold changes were computed as ratio, dividing growth rate of treated cells by their controls treated with DMSO. Mean value of growth fold change of all experiments ±standard error of the mean (SEM) was plotted. Two–way ANOVA with Sidak’s multiple comparisons test was performed with GraphPad Prism version 7.03 and yp, were computed as: yp = (y − T0)/(DMSO − T0), where y are the observed values. The model performance was estimated by a weighted standard error, and a weighted goodness-of-fit. Once the model was fitted, the drug 50% inhibitory concentration (IC50) could be estimated from inhibition rates by inverting the function . Mean value of IC50 of all experiments ±standard error (SE) was calculated. Student’s t tests were performed and P-values < 0.05 were considered significant. Mean value of senescence percentages of all experiments ±SE was plotted. Student’s t tests were performed and P-values < 0.05 were considered significant. Calculations and plots for IC50 and senescence were performed by R (version 3.3.1).For IC50, the signals were converted to proportions adjusting on time zero (T0) and on untreated DMEM control values and fitted using the 4-parameter logistic regression (R package nplr version 0.1–4). The proportion values, For GE data, the two-sided Wilcoxon–Whitney test was used to check for significant differences between two distributions. The distributions of PFS and OS were estimated using the Kaplan–Meier method and compared with the log-rank test.All codes used to analyze the data and generate the figures are available upon request.Supplementary material


The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines, namely; colon cancer HT-29 and triple negative breast cancer MDA-MB-468 cell lines. Also, their inhibitory activity against the cell surface expression of CD133 was examined. In particular, compound 4b emerged as a promising hit molecule as it manifested good antineoplastic potency against both tested cell lines , beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4c showed moderate activity against HT-29  and MDA-MB-468  cell lines, while they inhibited the CD133 expression by 14% and 48%, respectively. Finally, a single crystal X-ray diffraction was recorded for compound 4d.Cancer stem cells (CSCs) have been objects of intensive study since their identification in 1994. Adopting a structural rigidification approach, a novel series of 3-phenylthiazolo[3,2- Indeed, three main key properties of CSCs render them highly distinguishablePertaining to their close similarity to normal cells, it is problematic to segregate CSCs from non-CSCs within a tumour. But for the presence of surface cell antigens, the identification and separation of tumour initiating cells from more differentiated tumour cells would not have been possiblea]benzimidazoles  thio)-1-arylethan-1-ones . Chemical shifts are expressed in δ values (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques.Melting points were determined using a Gallenkamp melting point apparatus and are uncorrected. Infrared (IR) Spectra were recorded as KBr disks using the Perkin Elmer FT-IR (Fourier transform infrared) Spectrum BX apparatus. Mass spectra were measured on an Agilent Triple Quadrupole 6410 QQQ LC/MS (Liquid chromatography/Mass spectroscopy) equipped with an ESI (electrospray ionisation). NMR spectra were recorded on a Bruker NMR spectrometer. 1a–d (5 mmol) in glacial acetic acid (10 ml), 2-mercaptobenzimidazole 2  and conc. sulphuric acid (50 mmol) were added. The reaction mixture was heated under reflux for 2 h. The solid product obtained upon cooling was filtered off, washed with cold water then with petroleum ether, and recrystallised from ethanol to afford the corresponding sulphate salts 3a–d, respectively.To a solution of the appropriate acetophenone 3a–d (2 mmol) in water (10 ml), an aqueous solution of sodium bicarbonate was added. The reaction mixture was stirred at room temperature for 2 h. The solid formed was collected by filtration, washed with water, dried, and crystallised from ethanol to afford compounds 4a–d, respectively.To a suspension of the appropriate sulphate salts 1H NMR (DMSO-d6) δ ppm: 3.84 , 7.13 , 7.20–7.32 , 7.54 , 7.71 ; 13C NMR (DMSO-d6) δ ppm: 55.86 (OCH3), 109.23, 112.01, 114.68, 116.44, 119.23, 120.86, 121.47, 123.61, 130.61, 130.69, 133.66, 148.64, 157.03, 159.92; Anal. Calcd. for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99; Found C, 68.73; H, 4.28; N, 10.12.White crystals (yield 80%), m.p. 173–175 °C; 1H NMR (DMSO-d6) δ ppm: 3.82 , 6.99 , 7.09 , 7.12 , 7.27–7.31 , 7.69 , 9.67 ; 13C NMR (DMSO-d6) δ ppm: 56.23 (OCH3), 107.63, 112.07, 113.40, 116.14, 119.11, 120.06, 120.72, 122.38, 123.48, 130.22, 134.22, 148.17, 148.67, 148.77, 156.94; Anal. Calcd. for C16H12N2O2S: C, 64.85; H, 4.08; N, 9.45; Found C, 65.14; H, 4.02; N, 9.34.White crystals (yield 78%), m.p. 190–193 °C; 1H NMR (DMSO-d6) δ ppm: 3.79 , 3.83 , 7.07 , 7.22–7.38 , 7.71 ; 13C NMR (DMSO-d6) δ ppm: 56.62 (OCH3), 60.71 (OCH3), 106.95, 108.79, 112.23, 119.19, 120.89, 123.58, 124.64, 130.21, 133.83, 139.20, 148.68, 153.59, 156.93; Anal. Calcd. for C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23; Found C, 63.69; H, 4.69; N, 8.11.White crystals (yield 83%), m.p. 177–179 °C; 1H NMR (DMSO-d6) δ ppm: 5.28 , 6.73 , 7.22–7.38 , 7.59 , 7.68 ; Anal. Calcd. for C15H11N3S: C, 67.90; H, 4.18; N, 15.84; Found C, 68.15; H, 4.14; N, 15.73.White crystals (yield 75%), m.p. 185–186 °C; 4d were performed on a Bruker SMART APEX II D8 Venture diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 100 K. The structure was solved by direct method and refined with SHELXTL. E-maps provided the positions of all the non-H-atoms. The full-matrix least-squares refinement was carried out on F2’s using anisotropic temperature factors for all non-H-atoms. Crystallographic data for the structure reported in this paper have been deposited at the Cambridge Crystallographic Data Centre and allocated with the deposition number: CCDC 1429525.The measurements of the crystal of compound a]benzimidazoles 4a–d was evaluated at Stem Cell Therapy and Tissue Reengineering Program, King Faisal Specialized Hospital and Research Center, Riyadh, Saudi Arabia. In vitro anti-proliferative activity was measured by the cell growth inhibition assay. This assay was conducted by use WST-1(water soluble tetrazolium-1) reagent50 for each compound. HT-29 colon cancer cell line and MDA-MB-468 triple negative breast cancer cell line were purchased from the American Type Culture Collection. Cells were maintained in RPMI 1640 , supplemented with 10% FBS  , 100 IU/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine (Sigma). Cells were seeded into 96-well plates at 0.4 * 104/well and incubated overnight. The medium was replaced with fresh one containing the desired concentrations of the test compounds. After 48 h, 10 μl of the WST-1 reagent were added to each well and the plates were re-incubated for 4 h at 37 °C. The amount of formazan was quantified using ELISA (Enzyme Linked Immunosorbent Assay) reader at 450 nm.Anti-proliferative activity of the synthesised 3-phenylthiazolobenzimidazoles 4a–d, with 75–83% yield . The crystallographic data and hydrogen-bond geometry of 4d are presented in Supplementary data.Characterisation of compound a]benzimidazoles 4a–d was evaluated against human colon cancer cell line HT-29 and triple negative breast cancer cell line MDA-MB-468 using the WST-1 assay as described by Ngamwongsatit et al50) values. Among the tested compounds, compound 4b bearing a terminal phenyl ring substituted with two electron-donating groups proved to be the most potent against both cell lines; HT-29 and MDA-MB-468 with IC50 values of 9 and 12 μM, respectively. Compared to the positive control 5-FU , these results proclaimed compound 4b to be superior to the reference drug. Moreover, compounds 4a and 4c showed moderate activity against HT-29 cell line with IC50 values of 21 and 29 μM, respectively, which are comparable to that of 5-FU (IC50 = 15 μM). Luckily, both compounds evinced good antineoplastic potency against MDA-MB-468 cell line relative to 5-FU . Unfortunately, compound 4d bearing a p-amino phenyl group did not manifest any significant anti-proliferative activity against the cancer colon cell line HT-29 while it demonstrated moderate activity against triple negative breast cancer cell line MDA-MB-468 (53 μM)  .a]benzimidazoles 4a–d on cell surface expression of CD133 was evaluated at 10 μM via flow cytometry. The analysis was performed on a BD LSR II™ (BD Biosciences). The results expressed as a side population inhibition (%). Scrutinizing the obtained results disclosed that compound 4a bearing a m-methoy phenyl group only limitedly inhibited the CD133 by 13%, whereas compound 4b that possesses an extra electron donating group; an additional p-hydroxy group significantly inhibited the CD133 expression by 50%. Coherent to these findings, compound 4c that has three methoxy groups grafted on its terminal phenyl ring produced a comparable inhibition of CD133 (48%). These findings are in accordance with the previous conclusion that outlined the importance of incorporation of a lipophilic electron-donating group represented by a terminal phenyl ring substituted with a methoxy or a hydroxyl group. Regrettably, compound 4d containing a p-amino phenyl ring did not display any marked activity against the cell surface expression of CD133  based on structural rigidification of a series of 2-((benzimidazol-2-yl) thio)-1-arylethan-1-ones (Series 1) that proved to have antineoplastic activity and inhibitory activity of cell surface expression of CD133. The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines; colon cancer cell line HT-29 and triple negative breast cancer cell line MDA-MB-468. Moreover, their inhibitory activity against the cell surface expression of CD133 was determined in an attempt to explore their potential to eradicate CSCs as well as the tumour bulk cells. Compound 4b emerged as a promising hit molecule as it manifested excellent antineoplastic potency against both tested cell lines  beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4d exhibited good anti-proliferative activity against both cell lines and also significant inhibition potential of cell surface expression of CD133. On the other hand, structure of compound 4d was further substantiated via X-ray single crystal analysis.In conclusion, we designed and synthesised a novel series of 3-arylthiazolo[3,2-