Disruption of the reprogrammed energy management system of malignant cells is a prioritized goal of targeted cancer therapy. Two regulators of this system are the Fer kinase, and its cancer cell specific variant, FerT, both residing in subcellular compartments including the mitochondrial electron transport chain. Here, we show that a newly developed inhibitor of Fer and FerT, E260, selectively evokes metabolic stress in cancer cells by imposing mitochondrial dysfunction and deformation, and onset of energy-consuming autophagy which decreases the cellular ATP level. Notably, Fer was also found to associate with PARP-1 and E260 disrupted this association thereby leading to PARP-1 activation. The cooperative intervention with these metabolic pathways leads to energy crisis and necrotic death in malignant, but not in normal human cells, and to the suppression of tumors growth in vivo. Thus, E260 is a new anti-cancer agent which imposes metabolic stress and cellular death in cancer cells. The tyrosine-kinases Fer/FerT associate with the mitochondrial electron transport chain in cancer cells supporting their metabolic reprogramming. Here the authors discover a compound that disrupts Fer /FerT activity and selectively induces cell death of cancer cell lines displaying anti-tumor activity in vivo. To overcome these obstacles, a novel approach has been adopted based on targeting fundamental processes that characterize the reprogrammed metabolic and energy generation systems of cancer cells2. Specifically, while normal mammalian cells primarily utilize mitochondrial oxidative phosphorylation for adenosine-tri-phosphate (ATP) production, cancer cells remodel their glycolytic and mitochondrial machinery so that glycolysis is upregulated even under aerobic conditions, which would normally attenuate glycolysis, a phenomenon termed the “Warburg effect”3. The enhanced glycolytic capability of malignant cells might be related to the overexpression of glycolytic enzymes such as hexokinase II (HK II), which is present only at basal levels in normal somatic cells and can facilitate the malignant phenotype4. HK II bears a double catalytic domain and is attached to the outer mitochondrial surface via the voltage-dependent anion channel, thereby enabling it to directly and efficiently utilize mitochondria-produced ATP to phosphorylate glucose at a faster rate4. Although the “Warburg effect” is a hallmark of the reprogrammed metabolism of cancer cells, these cells remain dependent on the integrity and functionality of their mitochondria for ATP production and fatty acid synthesis, a requirement that becomes most profound upon transition of the malignant disease to a metastatic phase5. Thus, the mitochondrial machinery undergoes reprogramming during the development and progression of malignant disease, a change that is reflected in the altered activity of several key enzymes7.Targeted therapy of cancer is aimed towards the development of selective inhibitors of the aberrant and mutated regulatory pathways of tumor cells, thereby leading to the elimination of malignant tumors. However, vast amounts of accumulating evidence highlight the complexity and challenging nature of this goal. This complexity reflects the genomic instability of malignant cells, and their tendency to acquire resistance to therapeutic agents8 cells7. Fer populates several subcellular compartments in malignant cells, including the cytoplasmic membrane, mitochondria, and cell nucleus10. In the mitochondria, Fer and FerT associate with complex I of the mitochondrial electron transport chain (ETC) of malignant but not of normal somatic cells, thereby supporting ATP production in nutrient-deprived cancer cells, in a kinase dependent manner7. Furthermore, silencing of either Fer or FerT is sufficient to impair ETC complex I activity. Concomitantly, directed mitochondrial accumulation of FerT in nonmalignant NIH3T3 cells increases their ETC complex I activity, ATP production, and survival, contingent upon stress conditions imposed by nutrient and oxygen deprivation. Notably, enforced mitochondrial expression of FerT endowed the nonmalignant cells with an ability to form tumors in vivo7. Thus, recruitment of the meiotic FerT to cancer cell mitochondria highlights the primary role of reprogrammed mitochondria in tumorigenesis.A recently reported player in mitochondrial reprogramming in cancer cells is the intracellular tyrosine-kinase, Fer, and its sperm and cancer cell-specific truncated variant, FerT, which are harnessed to the reprogrammed mitochondria in colon carcinoma12 and its levels in malignant prostate tumors are significantly higher than those detected in benign growths/tumors13. Furthermore, downregulation of Fer impairs the proliferation of prostate, breast, and colon carcinoma8 cells10, induces death in CC and non-small cell lung cancer (NSCLC) cells15, abolishes the ability of prostate carcinoma PC3 and V-sis-transformed cells to form colonies in soft agar13, and delays the onset and reduces the proliferation rate of mammary gland tumors in HER2 overexpressing transgenic mice16. Fer was also shown to promote metastatic processes; downregulation of Fer prevents the metastatic spread of breast and lung adenocarcinoma tumors18. At the clinical level, high Fer expression levels have been linked to poor prognosis of hepatocellular-carcinoma (HCC)19, clear cell renal cell carcinoma21, postoperative NSCLC14, and high-grade basal/triple-negative breast cancer22. The above findings portray the Fer and FerT kinases, which share an identical kinase domain (KD)23, as potential targets for selectively affecting the reprogrammed metabolic systems of cancer cells.Several lines of evidence support the roles of Fer in the progression and growth of malignant tumors. The kinase was detected in all human malignant cell lines analyzed24. Autophagy governs controlled cellular self-degradation, which is important for balancing sources of energy at critical points in response to metabolic insults24. This process also plays a cell-preserving role by removing and recycling damaged organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes25. Though autophagy is considered to promote survival, it can also lead to a cellular death, like necrosis26. One of the drivers of autophagy and onset of necrotic death is the poly (ADP-ribose) polymerase-1 (PARP-1) enzyme. PARP-1 takes part in multiple cellular pathways, including the adenosine-mono-phosphate protein kinase (AMPK)–mammalian target of rapamycin (mTOR) signaling cascade27, which controls autophagy, and deregulated activity of PARP-1 can lead to ATP and NAD consumption and to the onset of necrotic death28.Although targeting cancer cell metabolism and mitochondria with synthetic compounds may prove useful, cancer cells can overcome metabolic insults by transiently inducing the autophagy salvage processIn this manuscript, we describe a novel synthetic Fer/FerT inhibitor, which compromises mitochondrial integrity, and activates PARP-1 and autophagy, thereby targeting the energy management system of cancer cells, and selectively leading to their necrotic death.29. To optimize this system for the identification and development of Fer inhibitors, we ectopically expressed Fer in the yeast strain BY4741-3702, which lacks the phosphatase encoding-ptc1gene. Ectopic expression of Fer was found to change the tyrosine-phosphorylation pattern of intracellular yeast proteins  system was adopted. This HTS system is based on the previously described growth inhibition of yeast cells by ectopic expression of a mammalian kinase in these cellsins Fig.  in a manins Fig. . Concomi 23 Fig. . Thus, aule Fig. . 0342 waule Fig. . New entule Fig.  and accoule Fig. . E260 anule Fig. .Fig. 1DeTo demonstrate that E260 directly targets Fer and FerT, an in vitro kinase assay was performed using a purified KD-containing fragment of these enzymes. This analysis demonstrated the direct inhibitory effect of E260 on this domain as reflected by the significantly decreased auto-phosphorylation level of the Fer/FerT KD when incubated with ATP and increasing concentrations of E260 Fig. . Moreove31, E260 exhibited inhibitory effects on the Fer-kinase activity as was reflected by suppressed auto-phosphorylation activity of the enzyme  of Fer by antibodies directed toward the N-terminal tail of the protein, it did eliminate the immunoprecipitation of Fer and FerT by antibodies directed toward the C-terminal-KD of both Fer forms Fig. , therebyyme Fig. . This suyme Fig. . Importayme Fig.  or when yme Fig. . To furtyme Fig. . Westernyme Fig. . In two yme Fig. . Howeveryme Fig. . The samyme Fig. . In compyme Fig. .32. To corroborate the lack of apoptosis in E260-induced cell death, the absence of apoptotic markers including cleaved caspase 3, cleaved PARP-1 and translocation of AIF to the nucleus33 was confirmed 37, or the necroptosis inhibitor—Necrostatin-1 (Nec-1)38, indicated that necroptosis takes part in the cellular death inflicted by E260, as seen by the attenuation of the E260 cytotoxic effect exerted by the combined treatment with Nec-139  analysis was performed on SW620 cells treated with E260. This revealed the deformation and loss of the normal elongated shape of mitochondria in cells treated with E260 for 16 h Fig. . Closer 4 h Fig. . The resDestruction of the mitochondria was accompanied by depolarization of the mitochondrial membrane potential (MMP) in E260-treated malignant cells Fig. . Notably40. In accordance with the observed energy deficit, the metabolic sensor AMPK was phosphorylated and activated41 in the E260-treated cells. This was accompanied by the inhibitory phosphorylation of the AMPK down-stream effector-Raptor on serines 792 and 722 and by a decrease in the activating phosphorylation of Akt . Surprisingly, inhibition of the E260-induced autophagy with 3-MA almost completely restored the ATP level in the E260-treated malignant cells  that contributes to the onset of death in the E260-treated CC cells. To identify cellular pathways in which Fer is engaged in malignant cells, and proteins that interact with this kinase, a proteomic, Fer-interactome analysis was carried out. One of the proteins that was repeatedly identified by this analysis was PARP-1 Fig. . Immunos44 Fig. , confirm45, we treated SW620 cells with E260 for 12 h, a time period that precedes cell death, and during which PARP-1 becomes activated. Cells were then subjected to terminal deoxynucleotidyl transferase (TUNEL) analysis46, which did not reveal any effect of E260 on DNA integrity in the treated CC cells  profile of E260 in mice. E260 exhibited a T1/2 of 175 min in the blood, and a volume of distribution of 4244 ml/kg suggesting an efficient distribution of the compound in the animal tissues50 Fig. . Based o50 Fig. . To eval50 Fig. . When th50 Fig.  correlat50 Fig. . To furt50 Fig. . Histopa50 Fig. . In cont50 Fig. . Importa50 Fig. . Accordi50 Fig. . Histopa50 Fig. , kidneys50 Fig. , and liv50 Fig. .Fig. 7Sy52. These unique metabolic pathways can be therefore exploited as selective targets for cancer intervention52. Moreover, recent studies suggest that when cancer cells progress toward aggressive and metastatic stages, their dependence on functional reprogrammed mitochondria becomes more prominent, thereby making the mitochondria an attractive target for cancer therapy53.Reprogrammed glycolytic and mitochondrial pathways are hallmarks of the altered energy generation system of malignant cells, which support the abnormal survival and proliferation needs of these cells7. In this study, we describe the development of a novel Fer and FerT inhibitor named E260, which inhibits phosphorylation activity of these kinases and exerts potent and selective anti-neoplastic effects. When incorporated into nanoscale micelles, E260 exhibited an EC50 of 400 nM and imposed a complete death of metastatic CC cells after 24 h of treatment. Interestingly, the high-grade metastatic CC and pancreatic cancer cells were more sensitive to E260 than low grade CC and pancreatic cancer cells, and no death was observed in treated normal human cells. Hence, while kinase inhibitors such as Staurosporine often exert broad range off-target inhibition, thereby demonstrating a non-selective cytotoxicity which eliminates both cancer and normal somatic cells55, E260 is a more selective agent. The selective death induced by E260 in malignant cells might be attributed to the specific association of Fer and FerT with the ETC complex I of cancer but not of normal somatic cells7. Accordingly, a selective inhibition of complex I was seen in the treated CC cells but not in normal cells. This coincides with the observed decrease in complex I activity in cells subjected to Fer-targeting or FerT-targeting siRNAs7, thereby supporting the inhibitory effect of E260 on the Fer and FerT activities in cells. Notably, mitochondria of E260-treated cancer cells underwent deformation and decrease in MMP, an effect that was not seen in normal cells. These imposed mitochondrial abnormalities are similar to the deleterious effects exerted by other ETC inhibitors such as rotenone, TTFA and chlorimipramine, which inhibit ETC complex I, II, and III activity, respectively58. Furthermore, cancer cells bearing severe mitochondrial respiration defects also exhibit deformed and swollen mitochondria, emphasizing the fact that mitochondrial integrity is compromised when the ETC function is impaired59. Concomitant with the mitochondrial deformation, E260-treated CC cells exhibited a decrease in ATP content of up to 50%, and this decrease did not occur in treated normal Hfb cells, further indicating the selectivity of E260. Low production of mitochondrial ATP can direct the onset of necrotic death32. However, cancer cells can maintain a balanced and compensating energy generation system by accelerating their glycolysis to a much higher rate than that occurring in normal somatic cells59. Indeed, CC cells treated with E260 exhibited an elevated glycolytic rate in their attempt to potentiate ATP production. This trend was further accelerated when the treated CC cells were allowed to grow under high glucose concentration, alleviating the decrease in ATP level, and delaying the onset of cell death.Fer and its truncated sperm and cancer cell-specific variant, FerT, are newly discovered components of the mitochondrial reprogrammed ETC of cancer cells25. A specific example of such a pathway is the complex I non-selective inhibitor, rotenone, which induces mitochondrial dysfunction leading to the activation of autophagy56. Similarly, selective complex I inhibition and onset of autophagy are evoked in E260-treated CC cells. E260 directed ATP deficiency led to the activation of AMPK, deactivation of mTOR and activation of autophagy60. However, despite of the induction of autophagy, almost all the mitochondria in the E260-treated CC cells were deformed and functionally impaired. This suggests the inability of the induced autophagy to recycle the severely damaged mitochondria and to salvage the energetically deprived cells. Moreover, the persistent activation of autophagy consumes ATP, thereby contributing to the energy crisis and onset of necrotic death in the E260-treated cells. By applying the necroptosis inhibitor-Nec-138 we further demonstrated the involvement of this programmed necrotic process in the death evoked by E260 in malignant cells39. The E260-induced autophagy and consequent necrosis seems also to be driven by a deregulated PARP-1, which is dissociated by E260 from its newly discovered regulator, Fer. Moreover, by excluding the possibility that E260 induces DNA damage, which would also activate PARP-1, we substantiate the notion that E260 leads to the activation of PARP-1 through its dissociation from Fer. PARP-1 supports autophagy through the AMPK–mTOR pathway, and its activity or knockdown can promote or prevent stress mediated autophagy, respectively27. We suggest that by acting simultaneously through at least two subcellular targets, mitochondria and PARP-1, of malignant but not of normal cells, E260 selectively imposes ATP deficiency, which is exacerbated by a continuously active process of autophagy. Although in this study we used 3AB as a PARP-1 inhibitor we cannot rule out the fact that 3AB may also inhibit other members in the PARP family, which could also contribute to the cellular effects shown in the current study. These combined and selective effects of E260 lead to the necrotic death of malignant cells without affecting normal cells. Importantly the potent and selective anti-cancer effect of E260 was also manifested in vivo.Interestingly, E260 did not cause a significant elevation in the glycolytic rate in normal human cells, reinforcing the fact that these cells do not need to compensate for ATP loss. Although the enhanced glycolysis delayed the E260-imposed death in CC cells, it did not prevent it. The limited effects of upregulated glycolysis might be linked to the downregulation of HK II, which accompanies the deformation of mitochondria by E260. Thus, E260 cooperatively impairs the functioning of two key energy generation processes in malignant cells, affecting both the mitochondrial and the glycolytic systems. Cells experiencing energy depletion and/or organelle damage can utilize the autophagy processes to maintain homeostasis and escape cellular deathCollectively, we describe here the development of a novel low molecular weight synthetic compound, E260, which targets Fer and FerT. By cooperatively activating energy-consuming processes and selective mitochondrial deformation in malignant cells, E260 is selectively lethal to cancer cells both in vitro and in vivo.Y715F23, respectively, and were ligated to the pAES426-expression vector (pAES). The Fer/FerT Kinase Domain (KD) (541–822 a.a) encoding fragment was cloned into the pRSFDuet-1 expression plasmid, and transformed into Escherichia coli BL-21, strain23.DNA fragments encoding native, HA-tagged Fer (Ha-Fer), and Ha-Fer bearing an inactivating mutation at the 715 position (Y715F) were cleaved from the previously constructed expression—plasmids PECE HA-Fer and PECE HA-FerThe following primers were used for Fer KD: Forward-5′-GCCGCGAATTCGAAATCAGGTGTAGTTCTGCT-3′ Backward- 5′-CGACTGCGGCCGCCTATGTGAGTTTTCTCTTGAT-3′7, unless stated otherwise.The following cell lines were used: Colon cancer cell lines: HCT116, HT29, RKO, SW620, SW48. Liver cancer cell line: Hep3B. Normal human cells: Hfb (human fibroblasts from human foreskin), CCD841CoN  and CD34 positive hematopoietic donor derived stem cells. Pancreatic cancer cells: PANC-1, SU.86.86. All cell lines were obtained from the American Type Culture Collection and grown in minimal essential medium (MEM), as described previouslyCells were authenticated using a morphology check by microscope and by characterization of the DNA profiles using short tandem repeat analysis.Cells were grown for 24 h in MEM  at 37°C with 5% CO2, and were then transferred for additional 24 h to anaerobic culture jar containing CO2-generating envelope . These conditions reduce the oxygen level in the jar to 1% within 30 mi.Saccharomyces cerevisiae BY4741-3702 (3702) strain cells bearing a deletion in the ptc1 gene, which encodes the PP3C phosphatase were grown in normal medium. Transformation of the various expression plasmids to the 3702 yeast cells and yeast growth post-transformation were carried out as previously described29. Yeast growth analyses were carried out by inoculating the cells into a 96-well plate with a starting optical density (OD) of 0.005 in each well. Each well was then treated with either dimethyl sulfoxide (DMSO) , 0342 dissolved in DMSO, or E260 dissolved in DMSO. The plates were shaken constantly and incubated for 60 h at 30 °C. OD in each well was determined at seven time points during incubation, at intervals of 10 h.Whole-cell lysates were prepared as follows: cells were collected and were then lysed with Tris buffer pH 7.5 lysis buffer containing: 20 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 2 mM sodium orthovanadate, 1% NP40, 1 mM NaF (sodium fluoride), 10 mM β-glycerophosphate, and supplemented with complete protease inhibitors cocktail (Complete mini Sigma 0469312400 ROCHE). In total, 30 μg protein lysates from each sample were resolved by 7.5% or 10% SDS–PAGE, and analyzed by western blotting. Protein bands were visualized using a horseradish peroxidase (HRP)-conjugated secondary antibody to rabbit or mouse IgG (Jackson) in conjunction with a WB chemiluminescence reagent (Pierce cat. 34080).All full length and un-cropped blots which correspond to the WB images that appear in this manuscript are available at Supplementary Fig. The following primary antibodies were used in various analyses: anti-HA tag , anti-actin , anti-LC3 , anti-HKI and anti-HKII , anti-pyruvate kinase isozymes M1/M2 , anti-phosphofructokinase-1 , anti-lactate dehydrogenase , anti-Tubulin , anti-poly [ADP-ribose] polymerase 1 (PARP-1) , anti-poly-ADP-ribosylation (PolyADPr) , anti-Fer , anti-Fer SH2 , anti-Fer N′ terminus , anti-Fer C′ terminus , anti-p-AMPK , and anti-phosphotyrosine , anti-mTOR , anti-p-mTOR , anti-Akt1/2/3 , anti-p-Akt1/2/3 , anti-Regulatory-associated protein of mTOR (Raptor) , anti-p-Raptor ser722 , anti-p-Raptor ser792 , anti-phosphoglycerate kinase 1 (PGK) , and anti-Caspase 3 .29. To adopt this system for the identification of Fer inhibitors, we ectopically expressed Fer in the yeast strain BY4741-3702, which lacks the phosphatase encoding-ptc1gene. This led to the attenuation of the yeast growth rate. Inhibition of the Fer-kinase activity was expected to relieve the yeast growth attenuation. This yeast-based HTS system was implemented in a Tecan multi-functional robot, and applied for screening a library comprised of 100,000 synthetic compounds . The robotic system seeded the yeast cells in 96-well plates and administered each of the different tested compounds at ascending concentrations ranging between 0.1 and 50 µM. The seeded yeast cells were then allowed to grow for 40 h, and OD600, was measured robotically by the ELISA reader installed in the system. Compounds which restored the growth of the Fer-expressing yeast cells by at least 30% were chosen, and their effective concentrations were determined in a second round of selection. The automated screening cycles followed by the selection of the effective compounds led to the identification of four hit compounds which demonstrated the highest restoration of growth profile of the Fer-expressing cells. These underwent an initial round of SAR analysis leading to the identification of two lead compounds, one of which was 0342.A yeast-based, HTS system was established for the development of Fer-kinase inhibitors. This HTS was based on the previously described yeast growth inhibition assay by a mammalian kinase ectopically expressed in these cells−5-carboxanilide (XTT) based viability assay. This viability assay was based on the same method of cell inoculation in a 96-well plates and administration of the different Fer-inhibiting compounds in ascending concentrations ranging between 0.1 and 50 µM. The seeded cells were incubated with the tested compounds for 96 h. Following incubation, XTT dye  was added according to the manufacturer’s protocol. Following incubation to allow uptake of XTT, the relative fluorescence intensities were measured in each well using an ELISA reader installed in the system. After complete data acquisition from the second HTS assay, we compared the results obtained from the two HTS systems and correlated the restoration of the yeast growth with the selective elimination of cancer cells. This cross analysis enabled us to successfully identify E260 as the best candidate for inhibiting the Fer-kinase activity and for selectively killing cancer cells. Notably, we found a direct correlation between the successful restoration of growth in Fer-expressing yeast cells and the effective and selective cytotoxic properties of the compounds in cancer cells. E260 was also computationally analyzed using the ChemSoft software  and proven to possess drug-likeness according to the Lipinski’s rule of 5 61.A second round of SAR based on the structure of 0342 was conducted, thereafter leading to the development and synthesis of E260. The HTS system excludes Pan-interfering compounds, un-selective toxic compounds, and compounds that are unable to penetrate the membrane of intact live cells. After identification of Fer-inhibiting compounds, we further tested the cytotoxic effect of these compounds on normal somatic and cancer cells. To this end, the Tecan robotic system was adjusted to conduct 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazoliumX in Supplementary Fig. 1H-NMR, HRMS, and LC–MS analyses  were determined using 1H-NMR, 13C-NMR, LC–MS, FTIR, and UPLC integrated with HRMS ; III ; IV ; Va ; VI ; IX ; and X . For X, a series of 2D spectra were also obtained , which allowed the full attribution of all carbon and proton signals and confirms the chemical structure as shown.NMR spectra were recorded, in Bruker instruments, for the following materials: LC–MS (HR) information: UPLC (Agilent 1260) integrated with HRMS were obtained on a 6545 QTOF instrument (Agilent) using the following parameters:HPLC-Agilent 1260: Column: Agilent Zorbax HD-C18, 1.8-Micron, 2.1 × 50 mm, mobile phase A: water/0.1% formic acid, mobile phase B: acetonitrile/0.1% formic acid, gradient: 15–95% B in 10 min, hold 95% B to 2 min, 95% B to 5% in 2 min, 5% B for 2 min, then stop. Flow rate: 0.5 ml/min. Column temperature: 30 °C. Sample preparation: dissolved in water/acetonitrile (1:1). Detector: 210, 254, and 355 nmHRMS parameters: Interface:. ESI (positive), SheathGasFlow 12, SheathGasTemp (°C) 400, Nebulizer (psig) 40, Gas Flow (l/min) 8, Gas Temp (°C) 300, OctopoleRFPeak 750, Skimmer1 45, Fragmentor 180, Nozzle Voltage (V) 0,VCap 3500.LC–MS information: HPLC-Shimadzu Analytical 10Avp series, autosampler: Gilson 215, ELSD: Sedex 75(55), mass spectrometer: PE SCIEX API 165(100). Column: Synergi 2 μ Hydro-RP Mercury, 20 × 2.0 mm. Mobile phase A: water/0.05% TFA. Mobile phase B: acetonitrile/0.05% TFA. Gradient: 0.01 min—5% B, 3.6 min—95% B, 4.5 min—95% B, 5.00 min—controller stop. Flow rate: 0.7 ml/min. Pressure range: 0–4500 psi. Sample preparation: dissolved in water/acetonitrile (1:1). Detector: 210, 254 nm. MS parameters: Interface: ESI (positive).Fourier transform infrared spectroscopy: FTIR spectrum was collected using a Thermo Scientific Nicolet iS10 FTIR spectrometer equipped with a Smart iTR attenuated total reflectance sampler containing a single bounce diamond crystal. Data were collected and analyzed using OMNIC software. Spectra were collected in the 650–4000 per cm range at a spectral resolution of 4.III was determined using the Buchi melting point apparatus M-560  set to default parameters. A quick capillary-thermometer-hotplate based method was used for all other compounds. Briefly, small amount of finely powdered crystals of the test compound was placed in a thin-walled capillary tube. The tube was placed in very close proximity to a thermometer and capillary tube was heated in a small electric hotplate  in a rate of heating no more than 1° or 2° rise per minute. The capillary was closely observed until the crystals were completely melted to determine the MP. Differences in measured versus literature reported MP values could result from differences in determination methods and compounds purity.Melting point determination: The melting point (MP) of Compound 1H-NMR (CDCl3) δ: 4.23–4.05 , 3.65 , 3.54 , 2.77 , 2.62 , 2.41 , 2.33 , 1.80–1.60 , 1.46 ; 13C-NMR (CDCl3) δ: 172.81 (C5′), 154.64 (C = O BOC), 79.48 (C-BOC), 55.38 (C8′′a), 54.74 (C8′′b), 45.99 (C10′′), 45.33 (C7′′a), 43.56 (C2′′a), 42.83(C2′′b), 41.61(C7′′b), 38.32 (C4′′), 28.42 (Me-BOC), 28.39 (C3′′); HRMS (m/z): [MNa]+ calcd. for C16H29N3O3, 334.21011; found, 334.20986. MP: 98 °C.1H-NMR (DMSO-d6) δ: 11.45 , 9.15 , 8.82 , 4.61–4.03 , 3.65–3.25 , 3.20–2.82 , 2.74 , 1.84–1.65 ; 13C-NMR (CD3OD) δ: 174.13 (C5′′), 54.51 (C8′′a), 54.20 (C8′′b), 44.27 (C2′′), 43.69 (C10′′), 43.65 (C7′′a), 40.06 (C7′′b), 36.30(C4′′), 26.65 (C3′′b), 26.43 (C3′′a); HRMS (m/z): [M + H]+ calcd. for C11H21N3O, 212.17574; found, 212.1762. MP: 298 °C.1H-NMR (DMSO-d6) δ: 3.30–2.92 , 2.90 , 2.39 , 2.35–2.17 , 2.15 , 2.05 , 1.57 , 1.51 , 0.92 ; 13C-NMR (CDCl3) δ: 65.28 (C5′′), 55.19 (C8′′), 53.64 (C7′′), 46.64 (C2′′), 46.08 (C10′′), 33.80 (C4′′), 32.30 (C3′′); HRMS (m/z): [MH]+ calcd. for C11H23N3, 198.19647; found, 198.19623.1H-NMR (DMSO-d6) δ: 8.55 , 7.18 ;); 13C-NMR (Acetone-d6) δ: 168.98 (C5), 143.16 (C2); HRMS (m/z): [M + H]+ calcd. for C2H3N3S, 102.01204; found, 102.01207. MP: 185–187 °C .1H-NMR (DMSO-d6) δ: 7.51 ; 13C-NMR (acetone-d6) δ: 171.57 (C5), 125.23 (C2); HRMS (m/z): [M + H]+ calcd. for C15H18BrNO, 179.92256 and 181.92042; found, 179.92289 and 181.92066 (1:1 doublet). MP: 192–193 °C .1H-NMR (DMSO-d6) δ: 8.68 , 7.77 , 7.28 , 2.90 , 1.22 ; 13C-NMR (CDCl3) δ: 149.32 (C4′), 145.39 (C6), 145.31 (C8), 133.82 (C2), 129.76 (C1′), 127.03 (C3′), 125.28 (C2′), 109.54 (C5), 33.95 (C5′), 23.90 (C6′) MS (ESI+) (m/z) [MH]+:calcd. for C13H12BrN3S 322.2, 323.9; found 322.2, 324.0; HRMS (m/z): [MH]+ calcd. for C13H12BrN3S, 322.00081; found, 322.00047. MP: 139–141 °C.1H-NMR (CDCl3) δ: 7.71 , 7.26 , 3.87 , 3.09 , 2.93 , 2.50 , 2.34 , 2.25 , 1.91 , 1.79 , 1.33 , 1.29 ; 13C-NMR (CDCl3) δ: 164.59 (C2), 147.50 (C4′), 143.56 (C6), 141.30 (C8), 132.09 (C1′), 126.64 (C3′), 124.57 (C2′), 108.58 (C5), 64.05 (C5′′), 55.15 (C8′′), 53.63 (C7′′), 48.45 (C2′′), 46.05 (C10′′), 33.83 (C5′), 33.15 (C4′), 29.76 (C3′), 23.98 (C6′); MS (ES+) (m/z): [MH]+ calcd. for C24H34N6S 439.0; found, 439.5;); HRMS (m/z): [MH]+ calcd. for C24H34N6S 439.26384; found, 439.26346. MP: 190–192 °C.1H-NMR (DMSO-d6) δ: 8.17 , 7.68 , 7.22 , 4.14 , 3.87 , 3.14 , 2.88 , 2.69  2.49 , 2.42 , 2.22 , 1.81 , 1.29 , 1.21 13C-NMR (DMSO-d6) δ: 173.36 (COOH), 164.14 (C2), 146.68 (C4′), 142.48 (C6), 140.40 (C8), 132.01 (|C1′), 126.29 (C3′), 124.14 (C2′), 109.35 (C5), 71.69 (CHOH-tartrate), 62.72 (C5′′), 53.75 (C8′′), 51.58 (C7′′), 44.13 (C10′′), 48.22 (C2′′), 33.03 (C5′), 32.07 (C4′′), 29.05 (C3′′), 23.74 (C6′); HRMS (m/z): [MH]+ calcd. for C24H34N6S,439.263; found, 439.263. MP: 206–207 °C.The following compounds were synthesized according to the scheme in Supplementary Fig. I were stirred with 218 mmol (35.36 g) of CDI in 500 ml chloroform for 1 h at room temperature. Then, 218 mmol (21.84 g) 1-methylpiperazine were added and the reaction mixture was stirred under reflux for 2 h. When the reaction mass was cooled down to RT, 200 ml water were added, phases were separated, organic phase was washed with water (2 × 100 ml), combined organic layers were dried under anhydrous Na2SO4, and the solvent was removed under reduced pressure to provide 66.55 g (98%) of compound II, as a colorless oil.218 mmol (50.00 g) of compound II were dissolved in 400 ml EtOH and 36 ml of concentrated hydrochloric acid were added. The reaction mass was then stirred under reflux for 2 h. When cooled down, the formed precipitate was removed by filtration, washed with EtOH and air dried to provide 36.40 g (59%) of compound III which was obtained as a white solid substance.217 mmol (67.57 g) of III were slowly added and the reaction mixture was refluxed for 48 h. When the reaction mixture was cooled down, 16 ml water were added drop wise, followed by the addition of 16 ml 15% water solution of NaOH and final addition of 48 ml water. The reaction mixture was then filtered and the solid product was washed twice with 250 ml THF. Solvent was removed under reduced pressure to provide 17.77 g (71%) of compound IV, which was obtained as a colorless oil.412.3 mmol (15.649 g) LiAlH4 were suspended in 450 ml of absolute THF, then 126.9 mmol (36.06 g) of compound V) were suspended in 50 ml of 85% formic acid in water solution, followed by the careful addition of 80 ml sulfuric acid. The reaction mixture was stirred for 3 h on a boiling water bath and was then left to cool down. 800 ml water were then added and the precipitate was filtered off, washed with water, and dried. A yield of 100 g (99%) 2-amino-1, 3, 4-thiadiazole (Va), was obtained.91.5 g (1 mol) of thiosemicarbazide (Va) and 156.07 g (1.902 moles) sodium acetate was prepared in 460 ml of glacial acetic acid. The mixture was stirred and 69.53 g. (0.435 mole) bromine, dissolved in 100 ml. glacial acetic acid, were added drop wise. The reaction mixture developed an orange color during the addition of the bromine.A suspension of 40.0 g (0.396 mole) 2-amino-1, 3, 4 –thiadiazole (VI).The reaction mixture was then heated for 1 h on a steam bath. At the end of this step, the reaction mixture was poured onto an ice cooled 2 L volume glass and the precipitated solid substance was recovered by filtration. The solid product was re-suspended in water and the mixture was adjusted to pH 11 with aqueous ammonium hydroxide. The obtained product was filtered off on a Buchner funnel, washed with water, and dried to yield 35 g (49%) 2-amino-5-bromo-1,3,4-thiadiazole (compound VI) were suspended in 180 ml absolute EtOH, then 70.5 mmol (17.0 g) 2-bromo-4′-isopropylacetophenone (VII)) were added and the reaction mixture was stirred overnight at RT. Then the mixture was stirred for 12 h at 60 °C, followed by additional stirring for 24 h at 78 °C under reflux. A fraction ( ~ 80 ml) of the solvent was removed under reduced pressure, to achieve a doubled concentration of the mixture. The product was filtered off, washed with 15 ml of cold EtOH, and dried to provide a pure compound VIII , which was obtained as a white solid substance.59.93 mmol (10.79 g) 2-amino-5-bromo-1,3,4-thiadiazole (VIII were suspended in 150 ml absolute isopropanol. Then 30.41 mmol (3.93 g) diisopropylethylamine (DIPEA), and 15.2 mmol (3.0 g) 1-methyl-4-(piperidin-4-ylmethyl) piperazine (IV) were added and the reaction mixture was heated at 85° C under reflux, overnight. After cooling down, the product was filtered off, washed with 5 ml EtOH, then washed twice with 15 ml acetone, and dried. This yielded 4.2 g (63 %) of compound IX which was obtained as a white solid substance.13.68 mmol (4.41 g) of compound IX in 150 ml EtOH, a hot (50 °C) solution comprised of 7.3 mmol (1.1 g) tartaric acid in 30 ml EtOH, was added with stirring. The reaction was then heated for 30 min at 78 °C under reflux. After cooling to RT the white precipitate was filtered off, washed with EtOH and dried. This yielded 4 g, ( > 95 %) of the product X which was obtained as white solid substance.To a hot (50 °C) solution containing 7.3 mmol (3.2 g) 2, 1 mM EGTA, 0.01% Brij-35) and 1 µM ATP . As a negative control, the KD protein was incubated in the same buffer without ATP. The KD and ATP containing mixture was incubated for 1 h at room temperature with ascending concentrations of E260 dissolved in DMSO or with DMSO alone. Following the incubation period, a sample from the incubated mixture was separated by SDS–PAGE and a WB analysis was performed using specific anti-Fer and anti-pY antibodies to evaluate the inhibitory effect of E260, as reflected by the diminished phosphorylation level of the Fer/FerT KD.The Fer/FerT KD protein fragment was expressed in bacterial cells as described above. It was then purified twice using the Talone cobalt affinity resin beads  and eluted using a 10-400 mM imidazole gradient. The protein was further purified using a 5000 Dalton cut-off dialysis and concentrated using a 10,000 Dalton cut-off Amicon Ultra-4 centrifugal filter . Following purification, a sample of the purified protein was separated onto SDS–PAGE and subjected to a WB analysis using specific antibodies directed towards the Fer/FerT KD, to verify the integrity of the purified protein. To test the effect of E260 on the Fer/FerT KD auto-phosphorylation activity, 0.5 µg of the Fer/FerT KD protein was incubated in 0.5 ml kinase activity buffer . For our analysis, we used the highest scoring pose (score = 7.8). Notably the second best pose (score = 7.3) was also positioned in the same cavity.Homology modeling of a Fer fragment extending from residues 447-820, including the enzymes SH2 and KD, was performed using the MODELER protocol in Discovery Studio version 4.0 . As a template, we used the structure of the corresponding domains in the Tyrosine-protein kinase Fes/Fps, PDB 3BKB chain A, with 66% identity, 80% similarity, and 1% gaps. Docking of E260 to Fer/FerT was performed using AutoDock Vina65 using the thermophoresis and Temperature Jump analysis. The dissociation constant (Kd) of the binding was calculated using nonlinear regression analysis program embedded in the device software.The Fer recombinant KD was produced and purified as described above and was labeled with a reactive dye NT-647 using N-hydroxy succinimideester chemistry using the manufacturer kit . E260 was dissolved in DMSO and incubated with NT-647-labeled Fer KD or DMSO in binding buffer  at concentrations ranging between 25 nM and 25 µM. The binding of E260 to the labeled Fer KD was measured by using the microscale thermophoresis technology in a Monolith NT.115 reader (NanoTemper) as detailed previouslyEmulsification of E260 was performed in Cremophor EL and PBS mixture. Photon Cross-correlation Spectroscopy measurements combined with Pade–Laplace analysis was carried out and the size distribution and stability of the micelles were determined. All analyses were carried out using NanoPhox .Whole-cell protein lysates  or nuclear proteins  were prepared from SW620 CC cells. These were taken for immunoprecipitation of Fer or FerT using specific antibodies as follows: Protein lysates (5 mg) were incubated overnight at 4 °C with 1:100 diluted antibodies. Antigen–antibody complexes were precipitated with protein A Sepharose  after 1.5 h incubation at 4 °C. Precipitates were washed twice with Tris buffer, pH 7.5, containing 20 mM Tris-HCl, 10% glycerol, EDTA 1 mM, 0.1% Triton X-100, 150 mM NaCl, and once with 75 mM NaCl. Recovered immunocomplexes were solubilized in Laemmli sample buffer and were separated on SDS–PAGE. The protein was visualized by western blotting using specific antibody.31.Deliberate activation of Fer which leads to its auto-phosphorylation was carried out as described by applying 16 h serum starvation followed by 20 min incubation of cells with 3 mM H2O2 Fer was immunoprecipitated from cells lysates (1 mg) for 2 h at room temperature using protein A Sepharose beads which were conjugated to antibodies directed toward Fer’s N′ terminus. The eluted precipitates were resolved on SDS–PAGE, and proteins were visualized by WB using specific antibodies.Cells death level was determined using the MultiTox-Fluor Multiplex Cytotoxicity Assay  according to the manufacturer’s protocol. Briefly, cells were inoculated into black 96-well plate. After 24 h, when the cells were completely attached, E260 or control solution were administrated at different concentrations and incubated for the desired period of time. Following the incubation period, the assay’s fluorophore which was used to determine the cell death levels was added to each well. The relative fluorescence intensity emitted by the fluorophore from each well was determined using an ELISA reader and was compared to the florescence intensity obtained from the standard curve drawn according to the manufacturer’s protocol to translate it to cell death percentage and normalized to the non-treated cells which were also used in each analysis.Concentration of the HMGB1 protein in the cells growing medium was measured according to the manufacturer’s instructions using the HMGB1 ELISA kit .7.Cell death was determined using the Annexin V and PI assay  as describedCells were grown in MEM or DMEM, washed twice with PBS and fixed for 5 min in Karnovsky fixative  on the tissue culture plate. Cells were then collected, and transferred to 1.5 ml tube for further 1 h fixation at room temperature. The cells were then stored O.N at 4 °C after which the samples were washed in 0.1 M cacodylate buffer and post-fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer  for 1 h. Samples were then dehydrated in alcohol and propylene oxide followed by embedding in Agar Mix. Thin sections (60 nm) were cut, stained with uranyl acetate and lead citrate, and observed in a FEI Tecnai transmission electron microscope.For microscopic and ELISA-based analyses of the MMP, tetra-methyl-rhodamine-ethyl ester incorporation assay was carried out according to the manufacturer’s instructions .7.Quantification of intracellular ATP was carried out using high pressure liquid chromatography (HPLC) as previously described7.Activity of the mitochondrial OXPHOS complex I (NADH dehydrogenase) was determined using the Microplate Assay for Human Complex I Activity (MS141-MitoSciences), according to the manufacturer’s instructions and as described7.Immunocytochemical analyses of cells treated with E260 or control solution were carried out as previously described9. Briefly, Fer was immunoprecipitated from SW620 CC cells, followed by protein separation on SDS–PAGE and LC–MS/MS analyses.Interactome analysis of Fer kinase was carried out as previously described7. The applied siRNA sequences were:siRNA-mediated silencing of Fer in SW620 cells was carried out using Lipofectamine2000 as previously described 1. siRNA-fer 5′-ACGUAUCCAAGUCUUGGCUACUUAU-3′ 2. siRNA-fer 5′-GGAAUUACGGUUACUGGAAACAGUA-3′ For negative control (siRNA-neg)–Stealth RNAi siRNA Negative Control Med GC  was used.6 cells were inoculated into a 6-well plate containing a cover slip, and fixed for 30 min with 4% paraformaldehyde following adhesion. The cells were then subjected to TUNEL analysis according to the manufacturer’s instructions .Samples containing 1 × 10All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Bar-Ilan University. The investigation of the pharmaco-kinetic properties of E260 was carried out using 30 ICR/CD-1 mice. Intraperitoneal (IP) administration was used, and the solution was administered as 10 ml per 1 kg of animal body weight.At 5, 15, 30, 60, 120, and 240 min after administration (five mice per each time point) mice were decapitated and blood samples were taken in EDTA-containing tubes. Plasma was prepared from the collected blood by centrifugation and then forwarded for LC–MS/MS analysis. A high sensitivity analytical method was developed for detection of E260. Analysis was performed in the Agilent 1100 HPLC system including degasser, the binary pump, autosampler, and absorption detector with variable wavelength ranging between 190 and 600 nm. The HPLC system was coupled with the tandem mass spectrometer API 2000 (Applied Biosystems). The TurboIonSpray ion source was used in the positive ion mode. Data acquisition was performed with Analyst 1.3.1 software.Cells were grown in MEM containing 1 g/l glucose or in DMEM with 4.5 g/l glucose. Secreted lactate, which correlates with the glycolytic rate, was determined using Lactate Colorimetric Assay Kit II according to the manufacturer’s protocol .6 SW620 or SW48 CC cells and divided randomly into experimental groups as previously described7. The mice were transferred from ad libitum diet to a stricter diet 2 days before inoculation to lower blood glucose levels. At this point the mice were also housed one per cage to ensure an even consumption of food. The diet was comprised of 3 g/mouse/day of standard chow, given at the same time every day. The food was consumed within an average time of 2 h, consequently the mice were kept without food for the next 22 h until the daily ration. The mice were kept on this diet throughout the experiment. The mice were randomized 4 days after tumor inoculation and placed again each in a cage. Mice were injected intraperitoneally every 12 h for 22 days with 25 or 50 mg/kg of the micellar E260 formulation, and control mice were injected with empty micelles.All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Bar-Ilan University. Nude mice  were inoculated with 1.5 × 1022. Blood samples were drawn by cardiac puncture. The blood was allowed to clot for 30 min at room temperature and serum separation was carried out by 20 min centrifugation at 1000 × g. The serum was analyzed immediately using the Cobas 6000-501 Chemistry Analyzer  according to the manufacturer’s protocols for each assay.Mice were sacrificed at day 22 of the xenograft experiments, as described above. Tissues from relevant organs and tumors were fixed in paraffin, sectioned (7 µm sections) and stained with hematoxylin and eosin stain for histopathological evaluation as previously describedOnset of necrosis was monitored using the Ethidium homo-dimer III (EthD III) incorporation assay, according to the manufacturer’s protocol . EthD III staining in necrotic cells or Hoechst staining in live intact cell were visualized immediately by Axioimager z1 fluorescent microscope .t-tests, with a P < 0.05 being considered significant. Results are depicted as mean ± standard deviation (±SD) or ±standard error (±SE) of the mean for n given samples. For statistical analysis of groups with multiple comparisons, one-way ANOVA analysis was performed with Bonferroni post hoc, with P < 0.05 considered significant.Statistical analysis was performed using the paired and unpaired Student’s The authors declare that all the other data supporting the findings of this study are available within the article and its Supplementary InformationPeer Review File


Sequence alignment and phylogenic analysis showed that strain JJ1 belonged to Pseudomonas, strain JJ2 belonged to Cupriavidus, strains JJ11 and JJ15 belonged to Bacillus, and strains JJ10 and JJ18 belonged to Acinetobacter. The tolerance capability of all the strains was the trend of Pb2+ > Zn2+ > Cu2+ > Cd2+, the maximum tolerance concentration to Cd2+ was 200 mg/L, to Pb2+ was 1600 mg/L, to Zn2+ was 600 mg/L, and to Cu2+ was 265 mg/L. Strains JJ1, JJ11, JJ15, and JJ18 could grow well under pH 9.0, and strains JJ2, JJ11, and JJ18 could grow well under 7% of NaCl. The results showed that as a whole these strains had high environmental adaptability. This is the first report that heavy-metal-tolerant bacteria were found from ramie rhizosphere soil, which could be as a foundation to discover the relationship between ramie, rhizosphere bacteria and heavy metals.Six bacteria strains from heavy-metal-polluted ramie rhizosphere soil were isolated through Cd Over exploitation for mineral resources has overloaded the endurance capability of the environment, the pollution caused by the release of heavy metals during mining is getting more and more serious Cheng . The heaCompared with physical and chemical methods, bio-restoration technology is gotten attention considerably because of its advantages of low cost, little harmfulness for environment, and high efficiency  Gaudich.) is a perennial plant with strong roots, which is easy to form a stationary rhizosphere environment. This crop can grow well in heavy-metal-polluted soils such as mining areas, heavy metals are accumulated mainly in its roots, stems, and leaves . The sequence results were uploaded into Genbank and compared with the reported sequences by BLAST alignment in NCBI. The sequences with high homology were selected to construct phylogenetic tree based on Neighbor-Joining Mega 6.0. The bootstrap was designed as 1000 times 2, 2.0 g/L ZnSO4, and 2.0 g/L CuSO4 were sterilized at 121 °C for 15 min, and aseptically added into the sterilized LB solid mediums to prepare the selective mediums containing 100, 150, 200, 250, 300 mg/L Cd2+, 1200, 1400, 1500, 1600, 1800 mg/L Pb2+, 130, 150, 185, 265, 300 mg/L Cu2+ and 200, 300, 400, 500, 600 mg/L Zn2+, respectively. The isolated strains were lined to inoculate in these selective mediums containing series of concentrations of heavy metals, culturing in incubator at 28 °C for 1–3 days. The healthy and well grown colonies were regarded as bacteria capable of resisting to the heavy metals.The initial solutions of 1.0 g/L CdCl600). The same dosage of bacteria was inoculated in the LB liquid mediums containing 1, 2, 3, 5, and 7% of NaCl , culturing under 28 °C, 250 r/min for 18 h in shaker. The OD values of bacteria suspensions with different salinities were determined with spectrophotometer under 600 nm (OD600).The pHs of LB liquid mediums were adjusted to pH 4.0, pH 5.5, pH 6.0, pH 7.0, and pH 9.0 with the solutions of 2 mol/L HCl and 2 mol/L NaOH. The activated bacteria were inoculated in the liquid mediums with the dose of 2% respectively, culturing under 28 °C, 250 r/min for 18 h in shaker. The OD values of bacteria suspensions with different pHs were determined with spectrophotometer under 600 nm . Strain JJ15 had the similarity of 99% with the model strain Bacillus salmalaya 139SI (KM051837.1). The 16S rRNA gene sequences of strains JJ1, JJ2, JJ10, and JJ18 had the maximum similarity with the strains of Chryseobacterium, Cupriavidus, Pseudomonas and Acinetobacter, respectively  and Cupriavidus taiwanensis KKU 2500-3 (JX962693.1), and had a closer genetic relationship with C. oxalaticus. Thus, strains JJ2 belonged to Cupriavidus. Strain JJ11 was grouped with Bacillus mycoides IARI-JR-40 (KF054993.1) and Bacillus anthracis JPR-02 (HE716942.1), and had a closer genetic relationship with B. mycoides. Strain JJ15 was grouped with Bacillus salmalaya 139SI (KM051837.1) and Bacillus cereus 165PP (KM349191.1), and had a closer genetic relationship with B. salmalaya. Thus, strains JJ11 and JJ15 belonged to Bacillus. Strain JJ1 had the maximum similarity with Chryseobacterium indoltheticum LMG 4025 (NR-042926.1), but was grouped and had a closer genetic relationship with Pseudomonas baetica PN4 (KC790260.1). Thus, strain JJ1 might belong to Pseudomonas. Strains JJ18 was grouped with Acinetobacter tjernbergiae Z2-S-TSA11 (KC213887.1). Strain JJ10 was grouped with strain 18 because of the closest distance showing on the phylogenetic tree, although strain JJ10 had the similarity of 99% with Pseudomonas helmanticensis OHA11T (HG940537). Thus, strains JJ10 and JJ18 might belong to Acinetobacter.The phylogenetic tree was shown as Fig. 2+ > Zn2+ > Cu2+ > Cd2+, the maximum tolerance concentration to Pb2+ was up to 1600 mg/L, to Zn2+ was up to 600 mg/L, to Cu2+ was up to 265 mg/L, and to Cd2+ was up to 200 mg/L. Relatively speaking, strains JJ1, JJ10, and JJ15 had stronger tolerance capability to heavy metals than the others.The whole trend of tolerance capability to heavy metals showed as PbThe growth diagram of the six bacteria under different pHs was shown as Fig. The growth diagram of the six strains was shown as Fig. Proteobacteria, two of them belonged to Firmicutes, and one of them belonged to Flavobacteria. Strain JJ2 had the same genus with Cupriavidus metallidurans which is the type strain of anti-heavy metal bacteria in soil (Lazzaro et al. Bacillus spp., was 99%, and these reported kinds of bacteria are very rich in the heavy-metal-polluted soils (Ellis et al. E. coli was as the tendency of Cd2+ 56 mg/L, Pb2+ 41.4 mg/L, Cu2+ 64 mg/L, and Zn2+ 65 mg/L (Nies E. coli. The highest tolerance to Cd2+ for strain No.9 originated from radiaiton-polluted soil was up to 2100 mg/L (Jing et al. Pseudomonas aeruginosa and Enterobacter cloacae isolated from Cd-polluted soil had the tolerance of 400 and 300 mg/L to Cd2+ respectively (Xiaoyan et al. Zhuzhou city in Hunan province is a heavy industry city, the soil near some mine refineries are polluted heavily by heavy metals such as Cu, Pb, Zn, and Cd, especially the pollution degree of Cd has been in a severity (Chaozhuang et al. g/L Nies . The resMicroorganisms which have the optimal pH 8.0, or between pH 9.0–10.0, are categorized into alkaliphiles. Microorganisms which have no optimal alkali pH but can grow well under alkali condition are categorized into alkalitolerants Yanhe . The sixTo sum up, this paper describes that heavy-metal-tolerant bacteria are first isolated from ramie rhizosphere soil polluted heavily with heavy metals. These six strains had a wide range of adaptability under different environmental conditions. Based on the tolerance capability to heavy metals, pH and salinity, strains JJ1, JJ11, JJ15, and JJ18 could be candidate materials for restoring heavy-metal-polluted soils with alkalinity. Strains JJ2, JJ11, and JJ18 could be used as candidate materials for restoring heavy-metal-polluted soils with high salinity. The metabolism of resistance to heavy metals for these bacterial strains is needed to be discovered further. The obtained results may lay a foundation for discovering the relationship between rhizospheric soil, microorganism, and ramie plant.


P < 0.05). All three generations of mice treated with swainsonine had significantly higher spleen, liver, and kidney indices and significantly lower body weights compared to the 0 mg/kg BW group (P < 0.05). For the first and second generations of treatment group, the copulation indices and the numbers of live pups on postnatal days (PND) 0, 4, and 15 were significantly decreased compared to those of the 0 mg/kg BW group (P < 0.05). The fertility and gestation indices of the treatment group of the first generation were significantly increased compared to the 2.50 mg/kg BW, 1.20 mg/kg BW, and 0.60 mg/kg BW groups of the second generation (P < 0.05). Cumulatively, these results indicate that swainsonine may cause reproductive and developmental toxicities in mice in both parents and offspring. Swainsonine is the primary toxin in locoweeds. It causes intention tremors, reproductive dysfunction, emaciation, and death. The objective of the present study was to evaluate the potential reproductive and developmental toxicities caused by swainsonine in mice. The treatment groups consisting of three generations of mice were given a range of concentrations of swainsonine by intraperitoneal injection (2.50 mg/kg body weight (BW), 1.20 mg/kg BW, 0.60 mg/kg BW, and 0 mg/kg BW). The 0 mg/kg BW group exhibited significantly fewer estrous cycles and an increased number of estrous ones compared to the 2.50 mg/kg BW, 1.20 mg/kg BW, and 0.60 mg/kg BW groups ( Astragalus and Oxytropis spp.), a taxa of the Legume family, are toxic plants in the western United States that frequently poison livestock  = 0.50).All extracts were collected using column chromatography, which was placed on the thin-layer plate using the capillary sample. The body and reproductive organ weights increased for each group during the estrus cycle, as reported in P < 0.05). Furthermore, the body weights of the 2.50 mg/kg BW group were significantly lower than those for the 1.20 mg/kg BW and 0.60 mg/kg BW groups at 7 and 15 days of treatment (P < 0.05).The body weights of the 2.50 mg/kg BW, 1.20 mg/kg BW, and 0.60 mg/kg BW groups were significantly lower than those of the 0 mg/kg BW group after 7 and 15 days of swainsonine treatment (P < 0.05). The ovarian index decreased from 0 mg/kg BW group to 2.50 mg/kg BW group.The ovarian index of the 2.50 mg/kg BW and 1.20 mg/kg BW groups of mice was significantly decreased compared to that of the 0.60 mg/kg BW and 0 mg/kg BW groups at 7 and 15 days of treatment. The ovarian index of the 0.60 mg/kg BW group was lower than that for the 0 mg/kg BW group (P < 0.05). The uterine index of the 2.50 mg/kg BW, 1.20 mg/kg BW, and 0.60 mg/kg BW groups was not significantly different from each other at 7 days of treatment (P > 0.05). However, the 2.50 mg/kg BW and 1.20 mg/kg BW mice displayed a uterine index lower than that of the 0.60 mg/kg BW and 0 mg/kg BW groups at 15 days of treatment (P < 0.05). The uterine index for the 0.60 mg/kg BW mice was also significantly lower than that of the 0 mg/kg BW group at 15 days of treatment (P < 0.05).The uterine index of the 2.50 mg/kg BW, 1.20 mg/kg BW, and 0.60 mg/kg BW groups of mice was significantly decreased compared to that of the 0 mg/kg BW group at 7 days of treatment (P < 0.05). The 0.60 mg/kg BW mice had significantly longer cycles compared to those of the 0 mg/kg BW group (P < 0.05). The estrous cycle length increased for the 2.50 mg/kg BW group compared to that of the 0 mg/kg BW group at 7 days of treatment. The 2.50 mg/kg BW group had significantly longer cycles compared to those of the 1.20 mg/kg BW, 0.60 mg/kg BW, and 0 mg/kg BW groups at 15 days of treatment (P < 0.05). The 1.20 mg/kg BW and 0.60 mg/kg BW groups also had longer cycles than those of the 0 mg/kg BW group, and there was a significant difference between the 1.20 mg/kg BW and 0.60 mg/kg BW groups at 15 days of treatment (P < 0.05).With respect to the length of the estrous cycle (in days), the 2.50 mg/kg BW and 1.20 mg/kg BW groups displayed longer cycles than those of the 0.60 mg/kg BW and 0 mg/kg BW groups at 7 days of treatment (P < 0.05). The number of mice undergoing estrous cycle in 0.60 mg/kg BW group was significantly fewer than that in the 0 mg/kg BW group (P < 0.05). This number also decreased from the 2.50 mg/kg BW group to 0 mg/kg BW group at 7 days of treatment. The 2.50 mg/kg BW group had significantly fewer mice experiencing estrous cycle than 1.20 mg/kg BW, 0.60 mg/kg BW, and 0 mg/kg BW groups at 15 days of treatment (P < 0.05). Finally, the 1.20 mg/kg BW and 0.60 mg/kg BW groups had fewer mice in estrous cycle than the 0 mg/kg BW group, and there was a significant difference between the 1.20 mg/kg BW and 0.60 mg/kg BW groups at 15 days of treatment (P < 0.05).The number of mice experiencing estrous cycle was lower in the 2.50 mg/kg BW and 1.20 mg/kg BW groups than in the 0.60 mg/kg BW and 0 mg/kg BW groups at 7 days of treatment (P > 0.05).The relative organ weights and body weights of the mice are shown in P < 0.05). In the F0 and F1 generations, the liver weights of the mice in groups I, II, and III were significantly higher compared to those of the group IV mice (P < 0.05). There was no significant difference among mice of the groups I, II, and III (P > 0.05). For the F2 generation, the F2-III and F2-IV mice had significantly lower liver weights compared to those of groups F2-III and F2-IV (P < 0.05); however, there was no significant difference between the F2-I and F2-II groups and between the F2-III and F2-IV groups (P > 0.05). The relative liver weights of the F1 generation were higher than those of the F0 generation, except for the group IV mice.For the relative liver weights of the F0, F1, and F2 generations, the experimental group had a significantly higher weight compared to that of the control group mice (P < 0.05); however, there was no significant difference among groups I, II, and III (P > 0.05). The spleen and kidney indices of the F1 generation were higher than those of the F0 generation, except for the group IV mice.For the spleen and relative kidney weights of the F0, F1, and F2 generations, the groups I, II, and III mice had significantly higher values compared to those of group IV (P < 0.05). However, in the F2 generation, mice in groups I, II, and III had significantly lower values compared to those of the group IV mice (P < 0.05).For the relative ovary and uterus weights of the F0 and F1 generations, groups I, II, and III had significantly higher values compared to the group IV mice (P < 0.05); however, there were no significant differences among groups I, II, and III (P > 0.05). The body weights of the F1 generation were lower than those of the F0 generation, except for the group IV mice.For body weights in the F0, F1, and F2 generations, groups I, II, and III were significantly lower compared to those of group IV (P < 0.05). The number of live pups on PND 4 for group IV was significantly higher compared to that of groups I, II, and III (P < 0.05). The number of live pups on PND 4 for group III was significantly higher compared to that of group I (P < 0.05). The numbers of live pups on PND 15 for groups III and IV were significantly higher compared to those of groups I and II (P < 0.05). The number of live pups on PND 15 for group IV was higher than that of group III (P < 0.05).The developmental findings are shown in P < 0.05). There was no significant difference observed among groups I, II, and III (P > 0.05).The body weights of the live mice in the F0 generation were higher than those for the F1 generation during the same period. For the F0 and F1 generations, the body weights of the live mice of groups I, II, and III were significantly lower compared to those of group IV on PND 0, 4, and 15 (P < 0.05). The copulation index of group III was significantly increased compared to that of groups I and II (P < 0.05). For the F0 and F1 generations, there were no significant differences among groups I, II, III, and IV with respect to gestation length (P > 0.05). The fertility and gestation indices of groups F0-I, F0-II, and F0-III were significantly higher compared to those of groups F1-I, F1-II, and F1-III (P < 0.05). There was no significant difference between F0-IV and F1-IV (P > 0.05).The reproductive findings are shown in  Oxytropis kansuensis. The cationic structure of swainsonine is similar to that of mannose, and its high affinity to mannosidase allows swainsonine to inhibit lysosomal α-mannosidase [In this study, we successfully isolated swainsonine fromnosidase . In animnosidase , 13. ThePrevious studies suggest that long-term ingestion of plants containing swainsonine induces significant toxic effects on reproduction in livestock . HoweverIn experiment 2, changes in organ weights were observed for the kidney, liver, and particularly the spleen in the F0, F1, and F2 generations. The kidneys displayed mean swainsonine concentrations of 154 ng/g when histologic lesions were first observed. Liver lesions were observed when the liver had a mean swainsonine concentration of 319 ng/g . The weiNatural and experimental long-term ingestion of locoweed causes serious dysfunction in the reproduction of livestock  in both males (mating behavior and libido) and females  . IngestiFeeding locoweed to pregnant ewes induces fetal cardiac dysfunction, delayed placentation, reduced placental and vascular development, hydrops amnii, abnormal cotyledonary development, interruption of fetal fluid balance, and abortion . We showIn conclusion, severe reproductive and developmental toxicities are associated with swainsonine poisoning. Specifically, swainsonine accumulation in the body prolonged the estrous cycle, decreased the number of live pups obtained on PND 0, 4, and 15, and reduced copulation and fertility indices in mice. Furthermore, the weights of the spleen significantly increased, and the body weights significantly decreased for mice in groups I, II, and III for the F0, F1, and F2 generations. Cumulatively, swainsonine toxicity significantly disrupts reproductive performance in the parent and offspring.


Neutral sphingomyelinase is known to be implicated in growth arrest, differentiation, proliferation, and apoptosis. Although previous studies have reported the involvement of neutral sphingomyelinase in hippocampus physiopathology, its behavior in the hippocampus during Parkinson's disease remains undetected. In this study, we show an upregulation of inducible nitric oxide synthase and a downregulation of neutral sphingomyelinase in the hippocampus of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced mouse model of Parkinson's disease. Moreover, the stimulation of neutral sphingomyelinase activity with vitamin 1,25-dihydroxyvitamin D3 reduces specifically saturated fatty acid sphingomyelin by making sphingomyelin a less rigid molecule that might influence neurite plasticity. The possible biological relevance of the increase of neutral sphingomyelinase in Parkinson's disease is discussed. Neuroinflammation was described to be involved in the pathogenesis of Parkinson's disease . It has Ten- to twelve-week-old male C57BL/6J mice weighing 25–30 g  were used as previously reported [Anti-nSMase and anti-NOS2 (M-19) were from Santa Cruz Biotechnology Inc. . SDS-PAGE molecular weight standard was from Bio-Rad Laboratories . VD3 was obtained from DBA Italia . Dulbecco's modified Eagle's medium (DMEM), bovine serum albumin, tetramethylrhodamineisothiocyanate-conjugated goat anti-rabbit IgG, and MPTP-HCl were from Sigma Chemical Co. . Lipid standards 16:0SM, 18:1SM, and 24:0SM were purchased from Avanti .n = 5) received MPTP under an acute protocol. Mice were given 4 i.p. injections of MPTP-HCl 2 hours apart and at a dose of 20 mg/kg (free-base). They were euthanized 7 days after the last MPTP injection. Control mice received an equivalent volume of 0.9% NaCl solution. Removed brains were postfixed overnight in fresh 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) solution, cryoprotected with 30% sucrose in 0.1 M PB and frozen in isopentane (−30°C). Brain free-floating sections (20 μm thick) encompassing the hippocampus were prepared using a freezing microtome . Sections were stored at −70°C until use.Animals were treated as previously reported . Groups w/v) bovine serum albumin (BSA) and 1% (w/v) glycine in PBS to block nonspecific sites as previously reported [w/v) BSA in PBS for 1 hr, washed three times in 0.1% (v/v) Tween-20 in PBS and two times in PBS, incubated with tetramethylrhodamineisothiocyanate-conjugated anti-rabbit IgG for 1 h, diluted 1 : 50 in 3% (w/v) BSA in PBS, and washed as above. The samples were mounted in 80% (w/v) glycerol, containing 0.02% (w/v) NaN3 and p-phenylenediamine (1 mg/ml) in PBS to prevent fluorescence fading. The antibody incubations were done in a humid chamber at room temperature. Fluorescent analysis was performed on a DMRB Leica epi-fluorescent microscope equipped with a digital camera.The cryostat sections were incubated overnight with 3%  were cultured as previously reported . VD3, diTotal protein concentration was evaluated spectrophotometrically at 750 nm by using albumin bovine serum as a standard, as previously reported .β-hexosaminidase, α-fucosidase, β-mannosidase, α-mannosidase, β-galactosidase, and β-glucocerebrosidase [14C–SM  was used as substrate. The reaction mixture contained 0.1 M Tris/HCl pH 7.6, 0.1 mM 14C–SM, 6 mM MgCl2, 0.1% Triton X-100, and 100 μg protein to a final volume of 0.1 ml. Incubations were performed at 37°C for 45 min. 2 ml chloroform and methanol (2 : 1) were used to stop the reaction, and 0.4 ml of 0.5% NaCl was added. Samples were centrifuged at 2000 rpm 10 min, and 0.5 ml of the upper phase was diluted in counting vials with 10 ml Ecoscint A and 1 ml distilled water; radioactivity was measured with a Packard liquid scintillation analyzer. The aSMase activity was measured as previously reported [14C–SM, 250 mM sodium acetate, 0.1% Triton X-100, and 100 μg protein of NFL or nuclei to a final volume of 0.1 ml. Conditions of incubation and radioactive analysis were performed as nSMase. The β-hexosaminidase, α-fucosidase, β-mannosidase, α-mannosidase, β-galactosidase, and β-glucocerebrosidase enzymatic activities were determined with the corresponding fluorogenic substrate and reaction buffer, as previously reported [Enzymes involved in nervous cell differentiation (nSMase)  and lysorosidase , 20 wererosidase . Brieflyreported . BrieflyLipids were extracted, and 16:0SM, 18:1SM, and 24:0SM standards were prepared as previously reported . Analysit-test was used for statistical analysis between control and experimental samples.Three experiments were performed for each analysis. Data are expressed as mean ± S.D., and in vivo situation of PD mice by using HN9.10e cells after having silenced nSMase expression. Physiological doses of VD3 [β-hexosaminidase, α-fucosidase, β-mannosidase, α-mannosidase, β-galactosidase, and β-glucocerebrosidase were quantified in untreated and VD3-treated HN9.10e cells. The activity of all these enzymes remained unchanged (Figures iNOS inflammatory cytokine has been reported to be highly expressed in striatum and substantia nigra of animals with PD . As the s of VD3  signific Figures . The exp Figures . To have Figures . Then weβ-hexosaminidase, α-fucosidase, β-mannosidase, α-mannosidase, β-galactosidase, and β-glucocerebrosidase in the VD3 response was consistent with observations suggesting the specificity of nSMase role in the hippocampus [SMase is an indispensable enzyme for the brain and plays an important role in signal transduction pathways by regulating the level of SM and ceramide species involved in physiological functions and pathological diseases. A wide variety of studies has noted the diversity of nSMase and aSMase functions during brain development/stem cell differentiation and ischemic/degenerative/stress responses , 27. Recpocampus , 29, 30.Notably, our results indicate that the increase of nSMase activity reduces specifically saturated fatty acids and consequently reduces the ratio saturated/unsaturated fatty acids of SM. So we think that the mechanism underlying the increase in unsaturated fatty acids is not dependent on a transformation of unsaturated to saturated fatty acids but on a specific action of nSMase on saturated SM. Since unsaturated fatty acids make the membrane more fluid , we hypoIn conclusion, for the first time, we demonstrate that in the hippocampus from PD-induced mice, the nSMase is strongly reduced in association with neuroinflammation and that the increase in nSMase activity following VD3 treatment in HN9.10e cells decreases specifically only saturated SM. Collectively, these results place nSMase as an essential enzyme for hippocampus function. Further investigation of the potential roles of nSMase in PD could provide a better understanding of the biological relevance of its level and activation in the hippocampus as well as the potential benefits of targeting nSMase with VD3.


Matrix metalloproteinase-2 protein expression in A549 and Calu-6 cells was increased by CML or pentosidine at 4–16 μmol/L. These two AGEs at 2–16 μmol/L enhanced nuclear factor κ-B (NF-κ B) p65 protein expression and p38 phosphorylation in A549 cells. However, CML or pentosidine at 4–16 μmol/L up-regulated NF-κB p65 and p-p38 protein expression in Calu-6 cells. These findings suggest that CML and pentosidine, by promoting the invasion, migration and production of associated factors, benefit NSCLC metastasis.Effects of carboxymethyllysine (CML) and pentosidine, two advanced glycation end-products (AGEs), upon invasion and migration in A549 and Calu-6 cells, two non-small cell lung cancer (NSCLC) cell lines were examined. CML or pentosidine at 1, 2, 4, 8 or 16 μmol/L were added into cells. Proliferation, invasion and migration were measured. CML or pentosidine at 4–16 μmol/L promoted invasion and migration in both cell lines, and increased the production of reactive oxygen species, tumor necrosis factor-α, interleukin-6 and transforming growth factor-β1. CML or pentosidine at 2–16 μmol/L up-regulated the protein expression of AGE receptor, p47 Advanced glycation end-products (AGEs) such as carboxymethyllysine (CML) and pentosidine are reactive compounds formed from glycosylation of sugars and macromolecules like proteins or lipids. CML and pentosidine could be endogenously synthesized under certain pathological conditions such as diabetes or Alzheimer’s disease, and these AGEs are considered as endogenous AGEs. Many foods including sauces, canned meats, nuts or grain products contain CML, pentosidine and other AGEs ,2. Thus,The studies of Weng et al.  and TsaoA549 and Calu-6 cells are human NSCLC cell lines, and have been widely used for NSCLC cell model researches . In our p > 0.05). However, CML or pentosidine at 4–16 μmol/L enhanced invasion and migration of A549 cells (p < 0.05). In Calu-6 cells, these two AGEs at 2–16 μmol/L promoted invasion and migration (p < 0.05).As shown in p < 0.05). Concentration-dependent manner was presented in raising TNF-α and TGF-β1 release in test cells (p < 0.05). CML at 4–16 μmol/L and pentosidine at 2–16 μmol/L up-regulated RAGE expression in A549 and Calu-6 cells (p < 0.05). In both cell lines, p47phox protein expression was enhanced by CML or pentosidine at 2–16 μmol/L (p < 0.05). However, CML or pentosidine at test concentrations did not alter gp91phox protein expression in two NSCLC cell lines (p > 0.05).CML at 4–16 μmol/L and pentosidine at 2–16 μmol/L increased the production of ROS, TNF-α, IL-6 and TGF-β1 in A549 and Calu-6 cells (p < 0.05). MMP-2 protein expression in A549 cells was increased by two AGEs at 4–16 μmol/L (p < 0.05). In Calu-6 cells, CML or pentosidine at 2–16 μmol/L enhanced MMP-2 protein expression (p < 0.05). CML and pentosidine at test concentrations did not change VEGF and MMP-9 protein expression in test NSCLC cell lines (p > 0.05).As shown in p < 0.05). These two AGEs at 1–16 μmol/L increased p38 phosphorylation in A549 cells (p < 0.05). In Calu-6 cells, CML and pentosidine promoted NF-κB p65 protein expression at 2–16 μmol/L and 4–16 μmol/L, respectively (p < 0.05). CML and pentosidine at 2–16 μmol/L enhanced p-p38 protein expression in Calu-6 cells (p < 0.05). CML and pentosidine at test concentrations failed to affect NF-κB p50, JNK and ERK1/2 protein expression or phosphorylation in test NSCLC cell lines (p > 0.05). As shown in p < 0.05).In A549 cells, CML or pentosidine at 2–16 μmol/L up-regulated NF-κB p65 protein expression  and pentosidine (90%) were purchased from Cayman Chemical Co. . Plates, medium, chemicals and antibiotics used for cell culture were purchased from Difco Laboratory . Human lung cancer cell lines, A549 and Calu-6, were obtained from American Type Culture Collection .2. The culture medium was changed every three days, and cells were subcultured once a week. A phosphate buffer saline  was added to adjust the cell number to 105/mL for various experiments and analyses. The plasma concentrations of CML and pentosidine in healthy people were in the range of 0.053–0.49 μmol/L [Cells were cultured in RPMI 1640 medium, containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin and 100 units/mL of streptomycin (pH 7.4) at 37 °C in 5% CO9 μmol/L . However9 μmol/L ,35. ThusCell proliferation was determined by using a bromodeoxyuridine ELISA colorimetric assay . Cells were counted by using a hemocytometer.5/100 μL) were seeded into the upper chamber in 200 μL of serum-free medium; and the lower chamber was filled with 0.66 mL of RPMI 1640 media containing 10% of FBS as a chemoattractant. After 6 h incubation for migration assay or 16 h incubation for invasion assay at 37 °C, the cells on the upper surface of the filter were removed by a cotton swab. The migrated or invaded cells to the lower surface of the filter were stained with 0.2% crystal violet in 10% ethanol. Four independent fields of invasive or migratory cells per well were photographed under the microscope to count the cell numbers. Data were calculated as a percentage of the control groups.Cell invasion and migration were measured in transwell chambers by matrigel- and fibronectin-coated polycarbonate filters, respectively. In brief, cells  was the difference in fluorescence values obtained between time 0 and 5 min. Results are expressed as RFU/mg protein. The levels of IL-6, TNF-α and TGF-β1 in cell culture supernatant were measured by ELISA kits . Protein concentration was determined by an assay kit , and bovine serum albumin was used as a standard.Cells were washed and suspended in RPMI 1640 medium. ROS level was determined by 2’,7’-dichlorofluorescein diacetate, an oxidation sensitive dye. Cells were incubated with 50 μmol/L dye for 30 min and washed twice with PBS. After centrifugation at 412× Nuclei pellets were isolated and re-suspended in a solution containing 20 mM HEPES, 1 mM EDTA, 0.4 M NaCl, 1 mM DTT and 25% glycerol. After incubation and centrifugation, supernatants were collected for protein concentration quantification by protein assay reagents . NF-κB p50/65 DNA binding activity was determined by an assay kit . The binding of activated NF-κB was processed by a primary polyclonal antibody against NF-κB p50/p65, and followed by treating with an antibody conjugated with horseradish peroxidase. 3,3′,5,5′-tetramethylbenzidine was the substrate. The absorbance at 450 nm was recorded. Data are shown as optical density (OD)/mg protein.phox, gp91phox, VEGF, ICAM-1, MMP-2, MMP-9, fibronectin, NF-κB (1:1000) or MAPK (1:2000)  at 4 °C overnight, and followed by treating samples with horseradish peroxidase-conjugated antibody for 3.5 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and the detected bands were quantified by normalizing against GAPDH.Cell was homogenized in protease-inhibitor cocktail containing 0.5% Triton X-100 . This homogenate was further mixed with a buffer composed of 60 mM Tris-HCl, 2% β-mercaptoethanol and 2% SDS (pH 7.2), and boiled for 5 min. Sample at 40 μg protein was applied to 10% SDS-PAGE, and further transferred to a nitrocellulose membrane  for 1 h. After blocking with a solution containing 5% skim milk for 1 h to prevent non-specific binding of antibody, membrane was reacted with monoclonal antibody against Bcl-2, Bax, caspase-3, caspase-8 (1:1000), RAGE (1:500), p47n = 10). Data were expressed as means ± standard deviation (SD), and processed for analysis of variance. Differences among means were determined by Fisher’s Least Significance Difference Test with significance defined at p < 0.05.The effect of each treatment was analyzed from 10 different preparations (CML and pentosidine enhanced invasion and migration of A549 and Calu-6 cells. These AGEs increased the production of ROS and inflammatory cytokines, and up-regulated protein expression of NADPH oxidase, RAGE, ICAM-1, fibronectin, MMP-2, NF-κB p65 and p-p38 in both NSCLC cell lines. These findings suggest that CML and pentosidine benefit NSCLC metastasis.