Incubation of ERα+ SKOV-3 cells with ERs agonist 17β-estradiol (E2) significantly enhanced sema 4D expression and rate of cell proliferation. Incubated with E2, ERβ+ SKOV-3 cells showed lower sema 4D expression and cell proliferation. Blocking ERα and ERβ activities with ICI182-780 inhibitor, sema 4D expressions and cell proliferation of ERα+ and ERβ+ SKOV-3 cells were recovered to control levels. Taken together, the data showed that sema 4D expression was positively correlated with the progress of ovarian cancer. ERα positively regulated sema 4D expression and accelerated cell proliferation. ERβ negatively regulated sema 4D expression and inhibited cell multiplication.Ovarian cancer is one of the most common malignancies in women. Semaphorin 4D (sema 4D) is involved in the progress of multiple cancers. In the presence of estrogen-like ligands, estrogen receptors (ERα and ERβ) participate in the progress of breast and ovarian cancers by transcriptional regulation. The aim of the study was to investigate the role of sema 4D and elucidate the regulatory pattern of ERα and ERβ on sema 4D expression in ovarian cancers. Sema 4D levels were up-regulated in ovarian cancer SKOV-3 cells. Patients with malignant ovarian cancers had significantly higher sema 4D levels than controls, suggesting an oncogene role of sema 4D in ovarian cancer. ERα expressions were up-regulated in SKOV-3 cells compared with normal ovarian IOSE80 epithelial cells. Conversely, down-regulation of ERβ was observed in SKOV-3 cells. Forced over-expression of ERα and ERβ in SKOV-3 cells was manipulated to establish ERα Ovarian cancer is the most common cause of death in women with gynecological malignancies, and it has the highest mortality rate among women in the world. Because of the lack of definitive early symptoms and efficient, specific and sensitive markers for ovarian cancer monitoring, the majority of patients diagnosed with late-stage ovarian cancer typically have a 5-year survival of 30% ,2. Prevein vitro and in vivo . RNase, DNase, and DNA marker (Takara) were purchased from Shanghai Bito Co. Ltd., China. Methanol, haematine, ICI182-780 (ERs inhibitor), 17β-estradiol (E2), and eosin were purchased from Sigma Co. (USA). Sixty normal ovarian tissues, 60 benign ovarian cancer tissues, and 60 malignant ovarian cancer tissues were obtained from Second Affiliated Hospital of Kunming Medical University. The malignancies were classified into early phase (I-II) and terminal stage (III-IV) on the basis of surgical-pathologic staging FIGO, 2006. All ovarian cancer tissues were histologically confirmed by two pathologists. Patients did not receive medication before surgery. The collection of human tissue samples was approved and supervised by the Ethics Committee of Kunming Medical University. Human ovarian cancer SKOV-3 cells and human normal ovarian IOSE80 epithelial cells were purchased from Yingrun Biological Co. Ltd., China. 293TA cell line was purchased from Funeng Biological Co. Ltd., China.2 and 95% air at 37°C.SKOV-3 and IOSE80 cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum . 293T cell lines were cultured in Dulbecco's Modified Eagle Medium  containing 10% (v/v) FBS, 100 units/mL of penicillin-streptomycin , and HOSE . All cell lines were cultured in a humidified incubator in an atmosphere of 5% CO-ΔΔCT method  according to the manufacturer's instructions. QRT-PCR was performed as previously described to assess the expression levels of sema 4D, ERα and ERβ using the 2T method . β-actin2. 293T-pLV-ERα, 293T-pLV-ERβ or blank plasmid were co-transfected SKOV-3 cells with Lenti-Pac HIV Expression Packaging Kit following the manufacturer's instruction (GeneCopoeia™). Forty-eight hours after, transfection efficacy was evaluated by inverted fluorescence microscope. The supernatant was harvested, filtered and cleared by centrifugation at 500 g for 10 min at 4°C. Three days after infection, 2 μg/mL puromycin was added to the culture media to select the cell populations infected with the lentivirus for 2 weeks. The expression of ERα or ERβ was detected by RT-PCR and western blotting in these three cell lines as described above. The cell lines transfected with 293T-pLV-ERα and 293T-pLV-ERβ and stably expressing ERα and ERβ were named ERα+ and ERβ+ SKOV-3 cells, respectively. Cells transfected with blank plasmid were named control (CK) SKOV-3 cells.ERα and ERβ highly expressed vectors were amplified from human genomic DNA and cloned into the XHO I and EcoRV site of the lentiviral vector pEZ-Lv105 . Viruses were packaged in 293T cells to generate 293T-pLV-ERα and 293T-pLV-ERβ lentiviral vector. SKOV-3 cells were cultured in 1640 medium with 10% FBS in a 37°C incubator with 5% COTissue samples were fixed in PBS containing 4% paraformaldehyde. The slide was deparaffinized in dimethylbenzene followed by rehydration in 80% ethanol. Then, 3% hydrogen peroxide solutions were added to the tissue slides to quench the endogenous peroxidase. After washing with the PBS three times, the slides were incubated with anti-sema 4D antibody  overnight at 4°C, then secondary antibody  were added and maintained for 2 h at room temperature. Finally, the slides were developed with 3,3-diaminobenzidine  and counterstained with hematoxylin. Images were obtained using an Aperio Scanscope in five randomized visual fields . Immunoreactivity for sema 4D was evaluated according to the numbers of positive cells and intensity of stained cells . The resTissue samples were fixed in PBS containing 4% paraformaldehyde. Then, the sections were deparaffinized in xylene and successively rehydrated with washes in 100, 95, 85, and 70% ethanol (2 min each). After that, slides were stained with hematoxylin (2 min) and rinsed with distilled water and 0.1% hydrochloric acid in 70% ethanol. The slide was then stained with 0.5% eosin for 2 min and rinsed again with distilled water. Finally, the slides were dehydrated with 95 and 100% ethanol successively, followed by xylene (3 min) and mounted with coverslips.+, ERβ+, and CK SKOV-3 cell lines were cultured in 6-well plates containing PRMI 1640 medium. When the density of cells was appropriately 80%, 2 mL E2 solution  were added to each well. After 24 h culture, the cells were harvested for sema 4D detection. For the ICI182-780 inhibitor treatment, a 100 nM ICI182-780 solution, which has been previously proved to disable ERs signaling efficiently, was added to the three cell lines cultured in PRMI 1640 medium in 6-well plates for 6 h  were added to each well and cultured for another 24 h.ERα for 6 h ,26. Afte+, ERβ+, and CK SKOV-3 cell lines with or without 100 nM ICI182-780 pre-incubation were seeded in a 96-well dish at a density of 5×103 cells per well and incubated in 1640 medium containing 10% FBS and 10–6 M E2. After 0, 6, 12, 18, 24, and 30 h, the cells were washed with PBS and incubated in 100 µL 1640 medium containing 10 µL Cell Counting Kit-8  solution for 120 min. The absorbance of each well at a wavelength of 450 nm was measured. Five duplicate wells were used for each measurement and experiments were repeated three times.ERαThe cells were lysed with RIPA lysis solution . After total proteins were extracted, a BCA protein assay kit  was used to quantify the proteins. Equal protein amounts were mixed with the 4× loading buffer  and then boiled for 5 min for protein denaturation. A total of 15 μg protein from each sample was loaded for 12% SDS–PAGE gel electrophoresis and then transferred to a polyvinylidene fluoride  membrane. Then, the membrane was incubated with Ponceau S staining solution for 2 min to judge the transfer efficiency of proteins. Once the proteins were proven to have transferred to the membrane successfully, the membrane was incubated with 5% fat-free milk for 30 min. Then, the membrane was incubated with anti-sema 4D, ERα, ERβ  or anti-β-actin  antibodies at 4°C overnight. Finally, the membrane was washed and incubated with corresponding HRP conjugated-secondary antibodies at room temperature for 2 h. The bands were visualized using an enhanced chemiluminescence system .t-test or, when more than two groups were compared, by one-way ANOVA, followed by Tukey's post hoc test. P<0.05 was considered to be significant.Data are reported as means±SE. The statistical significance of differences between the groups was assessed by Student's Sema 4D was highly expressed in ovarian cancer cells and tissues. To confirm the role of sema 4D in ovarian cancer, a total of 180 ovary samples including 60 benign ovarian cancer, malignant ovarian cancer and normal ovarian tissues each, were collected for the analysis of sema 4D protein expression by immunohistochemistry. These ovary samples were pathologically confirmed by HE staining . The resAs shown in + and ERβ+ SKOV-3 cell lines, respectively. Transfection efficiency was monitored with the inverted fluorescence microscope during the construction of cell lines. The two SKOV-3 cell lines transected with 293T-pLV-ERα and 293T-pLV-ERβ displayed green fluorescence  or expressed in a relatively low rate of 33.3%  in normal ovarian tissue and patients with ovarian benign tumors had a sema 4D expression rate of 58.3% . However, only 8.3%  of patients with ovarian malignancy negatively expressed sema 4D at an early pathological stage and patients at a late pathological stage showed higher sema 4D protein levels  than those in an early stage . Sema 4D expression was positively correlated with the progress of ovarian cancer. Likewise, we also found that sema 4D m-RNA and protein expression levels in ovarian cancer SKOV-3 cells were significantly higher than those in ISOE-80. A previous study by our group has reported that sema 4D expression was gradually enhanced with the progress of ovarian malignancy using immunohistochemistry. However, the association of sema 4D expression with the ovarian cancer grade was not well assured due to the small sample size used previously. Therefore, an expanded sample size of ovarian malignancy was utilized in the current study. Conclusively, the data further confirmed that sema 4D played an inductive factor in ovarian cancer as they function in other malignancies (Sema 4D has been reported to be up-regulated in several cancers , advancignancies .+ and ERβ+ SKOV-3 cells. ERα and ERβ did not directly affect the behavior of the cancer cells. They function by regulating a variety of targets in cancer cells in the presence of ligands. The two cell lines as well as control SKOV-3 cells were then incubated with natural ERs activator E2 and followed by detection of sema 4D expression. Q-RT PCR results showed that sema 4D m-RNA expression was enhanced in ERα+ SKOV-3 cells when treated with E2 solution. Correspondingly, the sema 4D protein was increased in ERα+ SKOV-3 cells. As expected, sema 4D expression levels were significantly decreased in ERβ+ SKOV-3 via E2 incubation. When cells were pre-incubated with ERs inhibitor followed by E2 incubation, sema 4D expression was not altered. The regulation of ERα and ERβ on sema 4D expression was completely abolished due to inhibition of ERs activity. In addition to regulation of sema 4D expression by ERα and ERβ, we also found that the cell proliferation of E2-incubated ERα+ SKOV-3 cells was faster than those of E2-incubated ERβ+ and CK SKOV-3 cells. The E2-incubated ERβ+ SKOV-3 cells multiplied even slower than CK SKOV-3 cells. Sema 4D up-regulation was directly attributed to the faster proliferation. The fact was further validated by pre-incubation of ICI182-780 inhibitor, which can remove the differentiation of both sema 4D expression and cell proliferation of E2-incubated ERα+, ERβ+ and CK SKOV-3 cell lines. The results suggested that oncogene property of ERα was possibly due to its positive regulation on sema 4D expression and acceleration of cell multiplication, whereas ERβ negatively controlled sema 4D expression and inhibited cell proliferation.ERα expression levels were significantly higher than ERβ in SKOV-3 cells. On the contrary, IESO-80 cells showed a significantly higher ERβ levels than those of ERα in the present study. These observations confirmed the fact that ERα is generally an oncogene and ERβ is an anti-oncogene ,29. SincERα and ERβ have been proven to show similar affinity to natural 17β-estradiol, which is the main female hormone secreted in ovary . ExcludiIn conclusion, the data presented here suggested that sema 4D was up-regulated and was an inductor in the progress of ovarian cancer. Sema 4D expression and cell proliferation of ovarian cancer was first proven to be regulated by ERα and ERβ in an opposite manner. Although the regulatory mechanism of ERs on sema 4D expression remains vague, the clues might help peers to explore it. A cautious use of estrogen drugs in MHT is advised.


Uncaria tomentosa was reported, the leaves being the plant part with the highest phenolic content and presenting the most heterogenous proanthocyanidin composition. Further, cytotoxicity of leaves extracts in two cancer cell lines was also found to be higher than in the remaining parts of the plant. In the present study, fractioning of U. tomentosa leaves polyphenolic extracts was performed using Diaion® HP-20 resin and a detailed characterization and quantification of fractions (n = 5) was achieved using advanced analytical techniques such as Ultra-Performance Liquid Chromatography coupled with Electrospray Ionization and Triple Quadrupole (TQD) Tandem Mass Spectrometry (UPLC/TQ-ESI-MS) and 13C-NMR. Oxygen Radical Absorbance Capacity (ORAC) and cytotoxicity on gastric adenocarcinoma AGS and colon adenocarcinoma SW20 cell lines were also determined in the different fractions. Results showed selective distribution of 32 non-flavonoid and flavonoid phenolics among the different fractions. ORAC varied between 3.2 and 11.8 μmol TE/mg in the different fractions, whereas IC50 of cytotoxicity on gastric adenocarcinoma AGS and colon adenocarcinoma SW20 cell lines best values were between 71.4 and 75.6 µg/mL. Fractions rich in proanthocyanidins also showed the highest bioactivity. In fact, significant positive correlation was found between total proanthocyanidins (TP) quantified by UPLC-DAD and ORAC (R2 = 0.970), whereas significant negative correlation was found between TP and cytotoxicity towards AGS (R2 = 0.820) and SW620 (R2 = 0.843) adenocarcinoma cell lines. Among proanthocyanidins, propelargonidin dimers were of particular interest, showing significant correlation with cytotoxic selectivity on both gastric AGS (R2 = 0.848) and colon SW620 (R2 = 0.883) adenocarcinoma cell lines. These results show further evidence of the bioactivity of U. tomentosa proanthocyanidin extracts and their potential health effects.In a previous study, the detailed low-molecular weight polyphenolic profile of the different plant parts  of  Proanthocyanidins, constituted by condensed flavan-3-ols units, have been the object of different studies due to their potential health benefits, mainly considering their antioxidant activity and the important role these may play by acting as cancer chemopreventive, anti-inflammatory agents, and by reducing risk of cardiovascular mortality . HoweverDespite the increasing number of studies on polyphenols and their anticancer effects, there is a general recognition of the need for further knowledge on the mechanisms and factors that influence these effects, such as the presence of direct antioxidant activity, the promotion of endogenous defenses against oxidative stress, epigenetic regulation by modulating microRNA (miRNA) and direct protein interaction . Some stUncaria tomentosa (Cat’s claw) extracts, including immunostimulant, anti-inflammatory, antioxidant properties, and protective effects against cancer  was the most abundant procyanidin in the different fractions, especially in LH-F3 (20.6% of PA) followed by B3 [(+)-catechin-(4α→8)-(+)-catechin], which also showed the highest proportion in fraction LH-F3 (10.6% of PA). Propelargonidin dimer at 4.47 min was the most abundant, especially in fraction LH-F3 (20.0% of PA), followed by propelargonidin dimer at 5.74 min, in particular in fraction LH-F4 (17.6% of PA).13C-NMR spectra for U. tomentosa extracts, to complement the characterization of polyphenols by UPLC-MS/MS [13C-NMR spectra measurements of the fractions obtained from U. tomentosa leaves. Fractions LH-F3 to LH-F7 showed signals characteristic of proanthocyanidins , δ 132.5 ppm (C1’ of the B ring) and δ 119.8 ppm (C6’ of the B ring). On the other hand, PP gave distinctive signals at δ 130.4 ppm (C1’ of the B ring) and at δ 129.4 ppm (C2’ & C6’ of the B ring). The cluster of peaks between δ 117–115 ppm were attributed to C2’, C5’ (PC) and C3’, C5’ (PP). trans (catechin and afzelechin units) and 2,3-cis (epicatechin and epiafzelechin units), respectively. Second, the confirmation of the formation of polymeric structures due to the broad peaks at δ 107.6 ppm (bonded monomer units C4µ→C8 and C4→C6), δ 73.4 ppm (C3 extended units) and δ 39–36 ppm (C4 extended units). The upfield shifts from B ring resonances  provided two main pieces of structural information. First, the stereochemistry of C ring, where the signals to δ 83.7 ppm and 77.1 ppm for C2 were assigned of both isomers, 2,3-13C-NMR spectra for the other fractions (LH-F4 to LH-F7) also presented structural information corresponding to the specific phenol content of these fractions  and PC (21.9%), that can be noted in the signals between δ 160 and 150 ppm. Fraction LH-F5 presented mainly signals characteristic for PP  whereas Fraction LH-F6 and LH-F7 showed mainly signals corresponding to flavalignan-cinchonains (FL), for instance at δ 135.5 ppm consistent with phenylpropanoid units.The ractions . For insU. tomentosa fractions along with the total proanthocyanidin (TP)  content for each fraction, obtained by UPLC/TQ-ESI-MS . A correlation analysis between ORAC and each phenolic group’s content was also performed and a significant positive correlation was found for propelargonidin dimers . At the individual level, a significative positive correlation with ORAC values  was found for three compounds, namely propelargonidin dimer at 4.47 min, procyanidin B2 and procyanidin B4.Q-ESI-MS . Among t50 values indicated that cytotoxicity of U. tomentosa extracts were dependent on the cancer cell line used and was also influenced by the phenolic composition of the different fractions. Fractions LH-F3, LH-F4 and LH-F5 showed the most promising results including important cytotoxicity in both cancer cell lines (IC50 < 130 µg/mL) and a selectivity index (SI) showing values greater than 3.5. As mentioned, selectivity could suggest an apoptotic effect directed towards cancer cells with respect to normal cells, which is important for any chemotherapeutic use [Cytotoxicity results are summarized in utic use ,22.U. tomentosa extracts LH-F3 and LH-F4 (IC50 > 500 µg/mL), it was affected by the remaining three extracts. Particularly, fraction LH-3 exhibited remarkable cytotoxicity on both adenocarcinoma cell lines  with highest selectivity, which is in agreement with the largest content of properlagonidins and procyanidins present in this fraction.The lower cytotoxicity shown by fractions LH-F6 and LH-F7 coincides with lower contents in procyanidin and propelargonidin oligomers observed in these fractions. In contrast, fractions LH-F3, LH-F4 and LH-F5 showing larger concentrations of these compounds also exhibited higher cytotoxicity towards both gastric AGS and colon SW620 adenocarcinoma cell lines. Whereas growth of normal Vero cells was not affected by R2 = 0.820) and colon SW620 adenocarcinoma (R2 = 0.843) (R2 = 0.786), but a significant correlation was not found for AGS cells (data not shown).A correlation analysis was carried out between total proanthocyanidins , polyphenolic groups contents (UPLC/TQ-ESI-MS), ORAC and cytotoxicity on both adenocarcinoma AGS and SW620 cell lines. A significant negative correlation was found between TP and both cancer cell lines, gastric AGS adenocarcinoma (= 0.843) , indicatR2 = 0.848) and colon SW620 adenocarcinoma (R2 = 0.883) (R2 = 0.785); whereas no correlation was found for SW620 cells.Finally, in relation to the selectivity of cytotoxicity towards cancer cell lines in respect to normal (vero) cells, the analysis indicated a significant positive correlation between the content of propelargonidin dimers and both cancer cell lines, gastric AGS adenocarcinoma (= 0.883) , suggest13C-NMR spectra allowed to confirm the presence of the main type of proanthocyanidin  found in this study by UPLC-MS/MS. In fact, 13C-NMR spectra for three different fractions  of U. tomentosa leaves. The fraction LH-F3 contained a significant amount of procyanidins (PC) (46%) and propelargonidins (PP) (37%), and, indeed, the assignment of distinctive signals for these compounds was clearly shown in 13C-NMR spectra, which allows corroborating the results delivered by UPLC-MS.R2 = 0.736) between total phenolic content and ORAC values for different plant extracts. Our results are more specific, indicating not only significant positive correlations with total proanthocyanidin contents (R2 = 0.970), but also with particular structures such as propelargonidin dimers (R2 = 0.790). Regarding cytotoxicity, the mechanisms underlying the effect of polyphenols extracts in some cancer cells remain to be elucidated, due to factors such as modulation of signaling pathways and epigenetic regulation of gene expression that are important modulators of cancer cell phenotypes, their effect in Reactive Oxygen Species (ROS) homeostasis, inhibition of mutagenesis, upregulation of mechanisms of DNA maintenance [U. tomentosa leaves, containing a richer mixture of proanthocyanidins dimers, are the ones more cytotoxic and also more selective towards both gastric AGS and colon SW620 adenocarcinoma cell lines with respect to normal vero cells. These results are in agreement with studies showing selective inhibition on the proliferation of breast cancer cell lines like MDA and MB-231 using an enriched fraction of proanthocyanidins [R2 = 0.786), while no correlation was found for SW620 colon cells. In sum, our findings are in agreement with polyphenols acting in a synergistic manner, and with the fact that potential selectivity on cells could be dependent on the structure of proanthocyanidins.In respect to bioactivity, the results of the evaluation of antioxidant capacity ORAC are in agreement with studies reporting a correlation between total polyphenolic contents and antioxidant activity . For insntenance ,27,28, antenance  and extrntenance . In factyanidins  or otherU. tomentosa. Using advanced analytical techniques such as UPLC/TQ-ESI-MS and 13C-NMR, results showed selective distribution of 32 non-flavonoid and flavonoid phenolics among the fractions, proanthocyanidin being predominant in those fractions with higher bioactivity. In fact, a significant positive correlation was found between proanthocyanidin contents and ORAC antioxidant capacity (R2 = 0.970), as well as significant negative correlations with cytotoxicity on AGS (R2 = 0.820) and SW620 (R2 = 0.843) adenocarcinoma cell lines. Among flavan-3-ols, propelargonidin dimers were of particular interest, showing a significant correlation with cytotoxic selectivity on both gastric AGS (R2 = 0.848) and colon SW620 (R2 = 0.883) adenocarcinoma cell lines. Considering that proanthocyanidins are metabolized by the gut by the action of microbiota, these results show the evidence of the potential health effects of U. tomentosa proanthocyanidin extracts on gut-related diseases, such as colon cancer [U. tomentosa, cytotoxicity tests should be carried out at equivalent proanthocyanidin concentration in the different fractions. According to these findings, further purification of valuable fractions to obtain other fractions or individual compounds would be of interest to develop novel commercial dietary ingredients or botanical drugs derived from U. tomentosa.This paper describes a successful method to obtain enriched polyphenolic fractions of n cancer . To furt


We conclude from these preclinical studies of telomerase as an oncotarget tested by imetelstat response that imetelstat has efficacy across the entire oncogenotype spectrum of NSCLC, continuous therapy is necessary to prevent telomere regrowth, and short telomeres appears to be the best treatment biomarker.Telomerase was evaluated as a therapeutic oncotarget by studying the efficacy of the telomerase inhibitor imetelstat in non-small cell lung cancer (NSCLC) cell lines to determine the range of response phenotypes and identify potential biomarkers of response. A panel of 63 NSCLC cell lines was studied for telomere length and imetelstat efficacy in inhibiting colony formation and no correlation was found with patient characteristics, tumor histology, and oncogenotypes. While there was no overall correlation between imetelstat efficacy with initial telomere length (ranging from 1.5 to 20 kb), the quartile of NSCLC lines with the shortest telomeres was more sensitive than the quartile with the longest telomeres. Continuous long-term treatment with imetelstat resulted in sustained telomerase inhibition, progressive telomere shortening and eventual growth inhibition in a telomere-length dependent manner. Cessation of imetelstat therapy before growth inhibition was followed by telomere regrowth. Likewise,  Lung cancer is the leading cause of cancer-related death in both men and women in the United States . Non-smaIn this regard, telomerase is expressed in 85-90% of all cancers including lung cancer and is responsible for the limitless replication potential of cancer . By contImetelstat (formerly GRN163L) is a telomerase inhibitor that binds the hTR RNA template component of telomerase preventing telomerase from elongating telomeres. Imetelstat is a synthetic lipid-conjugated 13-mer N3′P5′ thio-phosphoramidate  Supplem. PreviouIn the present study, we looked at telomere length and growth responses to imetelstat in a large panel of NSCLC cell lines representing many different oncogenotypes as well as a large spectrum of standard chemotherapy and targeted therapy response phenotypes. We conclude that: imetelstat has activity  across the oncogenotype and standard chemotherapy/targeted therapy spectrum of NSCLCs; imetelstat response phenotypes correlate with tumor telomere length and treatment duration; and surprisingly, we found no evidence of acquired resistance with long-term therapy. These results validate telomerase as a relevant target and suggest that selecting patients whose tumors have the shortest quartile of telomeres may serve as an enrollment biomarker for telomerase inhibitors.via Southern blot and ranged from 1.5 kb to 20 kb  and oncogenotypes (summarized in Cell lines were divided into quartiles based on telomere length . The firp > 0.05). By contrast, for HCC44, the most imetelstat sensitive line, the difference was highly significant (p < 0.0001). Thus, the response to imetelstat ranged from 96% inhibition in colony forming ability to no statistically significant response. The response phenotypes did not correlate with patient characteristics or oncogenotype. When response of the NSCLC lines to imetelstat colony formation was compared to telomere length, an overall direct correlation was not observed (p < 0.03)). Because there was minimal difference in telomere length between Q2, Q3 and Q4, these 3 quartiles were pooled and also show increased response compared to Q1  response phenotypes for 28 standard and targeted chemotherapies and a range of sensitivity is observed with these drugs. There was no correlation between imetelstat response phenotypes and NSCLC response phenotypes to other therapies  which represent a range of telomere lengths, patient characteristics and oncogenotypes . 1 μM imin vitro treatment (~180 population doublings) and cells were grown in the absence of drug. Cells were collected at 2, 4, 6 and 8 weeks after removal of imetelstat and telomere length assessed  and the shortest response-time to continuous imetelstat treatment. The growth rate slowed in less than 4 population doublings (11 days) and loss of replicative potential and concomitant decrease in the number of cells followed with continued treatment  in a subcutaneous xenograft model. Calu-3 tumor growth rate was significantly slower in the imetelstat treated group compared to control and final tumor weights were smaller  include adenocarcinoma, large cell and squamous cell carcinoma histology; Caucasian and African American patients; males and females ranging in age from 25 to 73; a range of oncogenotypes including different combinations of p53, KRAS, LKB1/STK11, and EGFR mutations , and botin vitro and in vivo. Calu-3, H1648, and HCC827 with initial telomere lengths of 1.5 kb, 2 kb, and 3 kb, respectively, all demonstrated slowed growth in vitro in a length dependent manner and showed reduced tumor burden in the presence of imetelstat in a xenograft model. H460  required more population doublings to reach a slowed growth and eventual cell death in vitro and showed no initial difference in tumor growth rate in the presence of imetelstat in vivo. However, with pre-treatment in vitro, even this originally non-responsive cell line shows a delay in tumor formation emphasizing the need for adequate imetelstat exposure. Taken together, these data support short telomeres as selection criteria for maximum response to imetelstat in patients.The mechanism of action of imetelstat dictates prolonged treatment is necessary to observe progressive telomere shortening with continued cell division and eventual senescence or cell death. This is accompanied by a lag phase with minimal phenotypic change other than decreased telomere length and suggests shorter telomeres would have faster response times. As expected, initial telomere length correlated with the number of population doublings and time required to achieve critically short telomeres required to trigger these effects both in vitro; surprisingly we saw no evidence for emerging resistance. To examine resistance in another way, we serial cloned H1648 in the presence of imetelstat, but once again saw no evidence of selection of resistant clones , all other cell lines were established in the lab of Drs. John Minna and Adi Gazdar at the NIH  or UT Southwestern Medical Center (referred to as HCCxxxx) (1981-2014) and are available through ATCC or the Hamon Center. Cells were cultured in RPMI with 5% fetal bovine serum in 5% COescribed  and was via the TRAPeze Telomerase Detection Kit (Millipore). 100,000 cells were resuspended in 100 μl 1x CHAPS lysis buffer. 1 μl (1000 cells) was used for the PCR reaction. For tumor samples, 5 μg of protein were used.Telomerase activity was measured using the telomerase repeat amplification protocol (TRAP) assay Telomere length was determined using a Southern blot protocol as previously described . Briefly500 cells were plated in triplicate in 6-well plates. The assay was terminated at 14-21 days when at least 50% of colonies had 50 or more cells per colony in control wells. Cells were fixed and stained with 6% glutaraldehyde and 0.5% crystal violet solution . ColonieSenescence associated β-galactosidase staining was performed as previously described .6 cells were injected subcutaneously into the right flank of 6-8 week old female NOD/SCID mice. Mice were randomized with 10 mice per group and treatment began 1 day after cell injection. 30 mg/kg imetelstat in saline or saline alone was given in 100 μL i.p. 3x/week [3 when tumors were harvested and weighed. For the pre-treatment studies, H460 cells were treated with 1 μM imetelstat for 12 weeks in vitro before injection and imetelstat treatment was not continued in vivo.Mouse work followed an IACUC approved protocol. 1×10 3x/week , 34, 35.


Listeriolysine-O (LLO) is a 50KDa protein responsible for Listeria monocytogenes pathogenicity. The structure of LLO (PDB ID: 4CDB) withdomains D1 to D4 is known. Therefore, it is of interest to identify conserved regions among LLO variants for destabilizing oligomerization(50 mer complex) of its monomers using appropriate inhibitors. Therefore, it is of interest to identify suitable inhibitors for inhibiting LLO.Previous reports suggest the use of flavanoids like compounds for inhibiting LLO. Our interest is to identify improved compounds todestabilize LLO oligomerization. We used a library (Zinc database) containing 200,000 drug-like compounds against LLO using moleculardocking based screening. This resulted in five hits that were further analyzed for pharmacological properties. The hit #1  was further refined using appropriate modifications for creating a suitable pharmacophore model LLO inhibition.The modified compound -penta-1,4-dien-3-one)shows fitting binding properties with LLO with no undesirable pharmacological properties such as toxicity. Listeria monocytogenes is known for its virulence among the mostfamous species of Listeria . NeverthKoster et al have revealed Crystal structure of LLO in in 2014.According to the crystal structure, it consists of four distinctdomains, which have been called D1 to D4 and each of them,playing a different role in LLO functionally. This study wasconducted to intend to illustrate some sequences of LLO, which aremore conserved in D1 to D4. Describing these sequences can be of agreat importance for modeling an inhibitor for inhibitingoligomerization of LLO monomers . Two conDuring past these years, some drug targets were introduced withbactericidal mechanism: Yasuhiro Gotoh et al. 2010), worked onspecific inhibitor against two component signal transductionsystems (TCSs) which could reduce virulence of bacteria withinhibiting the sensory domains of the sensors blocking the quorumsensing system . One yea10, workeThe X-ray crystal structure of 488 amino acids polymer of L.monocytogenes toxin (LLO) obtained from protein data bank(www.rcsb.org/pdb/home/home.do) with pdb access code of4CDB . The proPyRX software  was usedFAF Drugs 3 web server  was usedAmong 200.000 drug-like ligands, the 5 highest binding affinity hitswere selected for the further evaluation. These top 5 poses, whichindicated more negative binding affinity, were examined forpharmacokinetic analysis and rational drug design purpose. Theselected hits reached the binding affinity equal to -9.6 for hit 1, -9.4for hit 2, -9.1, -8.8 and -8.6 for hit 3, 4 and 5 respectively. Based onLipinski rule of 5 we analyzed the hits regarding pharmacologicalproperties. In In order to calculate the LD50 and probable nonspecific targets,PROTOX webserver was used. PROTOX prediction indicated LD50of RD-1 is 1170 mg/kg with toxicity class 4. This performanceoperated in average similarity 80.26% and prediction accuracy of78.26 %. Interestingly no toxicity target (nonspecific) was found byPROTOX as well. As FAFDrugs and PROTOX indicated, the RD-1is a lead compound and can serve as a new drug to inhibit LLO.Identification of an LLO inhibitor to destabilize its 50-meroligomerization is of interest. We describe the identification,modification and design of an LLO inhibitor named RD-1 withfitting properties for further consideration. It should be noted thatfurther in vitro studies are needed to confirm binding andinhibition of LLO.


High-throughput screening (HTS) strategies and protocols have undergone significant development in the last decade. It is now possible to screen hundreds of thousands of compounds, each exploring multiple biological phenotypes and parameters, against various cell lines or model systems in a single setting. However, given the vast amount of data such studies generate, the fact that they use multiple reagents, and are often technician-intensive, questions have been raised about the variability, reliability and reproducibility of HTS results. Assessments of the impact of the multiple factors in HTS studies could arguably lead to more compelling insights into the robustness of the results of a particular screen, as well as the overall quality of the study. We leveraged classical, yet highly flexible, analysis of variance (ANOVA)-based linear models to explore how different factors contribute to the variation observed in a screening study of four different melanoma cell lines and 120 drugs over nine dosages studied in two independent academic laboratories. We find that factors such as plate effects, appropriate dosing ranges, and to a lesser extent, the laboratory performing the screen, are significant predictors of variation in drug responses across the cell lines. Further, we show that when sources of variation are quantified and controlled for, they contextualize claims of inconsistencies and reveal the overall quality of the HTS studies performed at each participating laboratory. In the context of the broader screening study, we show that our analysis can also elucidate the robust effects of drugs, even those within specific cell lines. Caenorhabditis elegans [High-throughput screening (HTS) strategies allow researchers to assess the effects of thousands of compounds on drug responses in one large experimental setting. Over the past decade, applications of HTS for drug discovery and drug effect characterization studies have steadily increased, ranging from studies focusing on the assessment of multiple phenotypic endpoints in high-content screening , the eva elegans  as well  elegans . HTS stu elegans , the Can elegans  (CCLE) a elegans  (GDSC) – elegans  has expl elegans  (CCMI) a elegans , 10.Assessing the reliability of HTS studies is not trivial given the number of compounds typically considered, the number of reagents used, the way in which constructs such as plating schemes and distributed robotic handlers are set up, the manner in which dose response curves are constructed, and the fact that different labs likely follow slightly, if not overtly, different protocols; i.e., the sources of HTS cells, tissues or organisms, cell culture and assay conditions, reagents, consumables and instrumentation are not standardized within the community. This is particularly true for, e.g., tumor-derived cell line-based screening studies where the nature of the source cell lines, their procurement and sustenance as well as the responses measured on them may vary widely between different laboratories. In addition, although diverse in execution, many cancer-oriented HTS studies focus on cell counts upon stimulation with a drug that reflect that drug's ability to kill cells derived from a specific cell lines across differing drug concentrations. These concentrations often range from inducing no response  to a very strong response . The dose ranges necessary to achieve variation in the number of cells killed and establish a dose response curve are very hard to anticipate, often leading to different labs using different concentrations and numbers of concentrations.50) is then extracted from these curves [50 values, which are often coupled with related response measures such as the area under each dose response curve (AUC), are then used to determine the sensitivity or resistance of each cell line to the different drugs. Recently, the IC50 and AUC results from two large cancer cell line HTS studies, the Cancer Cell Line Encyclopedia [After having established the drug concentrations or doses to be used and applying them to cells derived from a single cancer cell line, sigmoidal curves are often fit to the cell counts associated with each drug dose to generate drug-specific dose response curves (DRCs). This is repeated for each cell line. The half minimal inhibitory concentration  aclopedia  (GDSC) sclopedia , 8. The clopedia , 8, 13. clopedia .In order to assess the reliability of HTS data, we conducted a study of intra- and inter-site experimental variability across melanoma cell lines treated with 120 different drugs that are either in use in clinical trials or have been FDA-approved for use in treating cancers. Our study was motivated by not only the controversies surrounding the reliability of the CCLE and GDSC data sets, but also by our engagement in a large clinical trial exploring the utility of personalized treatment for late-stage BRAF wild-type melanoma . We firsTo analyze the data produced from the two nine-concentration HTS studies, and to assess the variability of the results, we used flexible linear models and analysis of variance (ANOVA) techniques. These traditional techniques allowed us to examine how variation in drug responses  is impacted by different factors, such as the laboratory, the drug used, the plate on which specific assays were conducted. We also considered interaction terms in the models . We chose not to generate dose-response curves and extract IC50 values for use in our analyses, since our interests were in quantifying as many sources of variation as possible and not condensing or obscuring any of them in dose-response relationships reflected in single derived value. Thus, we modeled the dosage effects of each drug as a separate independent or explanatory variable for drug response variation. It is well-known that models that are “saturated” in that they exhaustively model the effects of independent variables and their interactions are inherently linear. This fact is exploited in many contexts, most notably econometrics, to help draw causal inferences between independent and dependent variables . Althougp-value < 2.2e-16 for drug responses for each drug) and that the non-parametric Spearman correlation coefficient, rather than the standard Pearson correlation coefficient, would be more appropriate for use in assessing the consistency between the two data sets. We computed pairwise Spearman correlations for the replicates at 0.1, 1.0, and 10.0 μM concentrations across each drug and cell line , we also calculated the Spearman correlation coefficient between the SBP and TGen response data for each of the nine concentrations and also across all nine concentrations . Individual pairwise scatterplots for each dose across the 46 drugs indicate that for a large number of them, there is a high degree of between-laboratory consistency. As in the three-concentration drug response analysis performed, the drug responses were more consistent at higher doses and across all dosages when considered together  was highly significant. Furthermore, only one SBP plate yielded a more significant t-statistic (plate 27) than the TGen plate 25 . As expep-values; in fact many more than would be expected by chance alone. These results indicate that although drugs may influence variation in cell viability across the experiment as a whole, some of the cell viability variation may be laboratory-specific  while considering other factors, we added interaction terms to the linear models. When we accounted for laboratory x drug interactions, we found that many of the drugs exhibited significant interaction term specific . 19 drugs Figure , indicats Figure . As expes Figure . ImportaThe consideration of three-way interaction effects led to additional insights into the factors contributing to cell viability in a pronounced enough way to rise above the “noise” in cell viability across the HTS experiments as a whole. Laboratory × drug x dose effects explained 2.5% of cell viability variance and drug x dose x cell line explained 3.6% of the variance . These fTo investigate whether the use of an arbitrary drug as the comparator drug impacted our initial analyses in any way, we set out to identify a group of cell line and drug combinations that did not exhibit any dose-response or general drug effect. These combinations would then effectively act as a group of controls whose cell viabilities represent simple noise and technical variation when compared to the other cell line-drug combinations. These analyses would allow us to see if compelling drug or drug x cell line effects can be identified that rise above the noise and variation exhibited in the HTS experiment as a whole. As noted in the Methods section, we fit a three-parameter sigmoidal, a four-parameter sigmoidal, a linear, and a constant model to each of the cell lines for each drugs and dosages to characterize dose response curves. We then selected the drug-cell line pairs where the constant model fit the data the best based on the Akaike Information Criterion (AIC), since this would be indicative of no evidence of a drug or dose-dependent response. The set of drug x cell line combinations that suggested no evidence for a drug or dose-dependent response was then used as the comparator group in a re-analysis of the cell viability data. The re-analysis suggested that the laboratory in which the assays were done explained a minimal proportion of the variance (0.03%). However, drug effects explained 41.2%, dose 5.24%, cell line 4.94%, and plates 3.23% of the variation in cell viability across the experiment . Interesp-values surpassed Bonferroni-adjusted significance thresholds  effects in the model, we found 54 significant drugs with drug-effect resholds . When thresholds . Strikinresholds .50 values provided in the CCLE and GDSC data sets along with our SBP and TGen data on the four cell lines and six common drugs they had in common. In these models, we randomly selected CCLE as the comparator laboratory, the drug Crizotinib as the comparator drug, and cell line A375 as the comparator cell line. Similar to the analysis comparing the raw SBP and TGen data, we found that laboratories were not significant predictors of IC50 values when we also accommodated drug, cell line, and laboratory effects in the model. We note, however, that we did observe some laboratory-specific effects for one of the six drugs, Nilotinib  and hence the DRCs. In this context, once outlying plates have been identified, it is possible to reassess the reliability of the HTS data using the traditional methods while excluding those outlying plates . Additionally, ANOVA methodology allows for the simultaneous analysis of all cell viability data across all drugs to identify true and very compelling signals that have been obtained in a HTS experiment. Although the specific plating scheme in our experiments  for each cell line for each drug and then compare, e.g., ICs Figure  allowed in vitro drug screening can shed light on the potential for personalized medicine [In terms of specific findings, we observed that laboratory effects only explained a small fraction (0.03%) of the cell viability variation in our HTS setting. This contrasts with the results of studies described in recent publications about the reliability of the data associated with two very large cancer cell line screens pursued by different groups at different sites , 6, 8, 9medicine . We alsoUltimately, assessing the variability, reliability and reproducibility of HTS data by designing the study to contrast different experimental conditions could add to the overall experimental costs. As a result, alternative methods that can account for sources of variation are needed. Our analysis at the very least highlights the importance of using consistent dosing and plating schemes that include specific controls, which would allow researchers to not only measure and test for the impact of experimental factors on the outcomes, but also adjust for these specific experimental effects when making claims about, e.g., drug and cell-line specific effects. Thus, ANOVA-like methods can accommodate different experimental factors that may influence the HTS assays in pronounced ways and also reveal compelling signals attributable to drugs and experimental compounds. Although our analysis was performed only within the context of melanoma, and, importantly, only within the context of four melanoma cell lines and 120 drugs, our overall ANOVA-based approach can be used to reassess the quality of publicly available data as well as additional HTS data.We initially performed a nine-concentration  HTS study  on 40 melanoma cell lines across 120 drugs  measures were obtained post drug administration and were normalized to the plate-specific average DMSO cell:We also pursued an independent three-concentration HTS study  at SBP across 30 melanoma cell lines (29 in common with the nine-point screen) on all 120 drugs as described above. Finally, we performed the primary nine-concentration HTS study across all 120 drugs for four melanoma cell lines in common with the initial nine-point SBP screen  at the Translational Genomics Research Institute (TGen). For the TGen HTS study, the experimental protocols were similar to the SBP screen, with minor differences. Specifically, the compounds were pre-spotted to white, solid-bottom 384-well assay plates (Greiner Bio-One) using ATS . Additionally, prior to measuring the luminescence using an Analyst GT plate reader , 25 uL CellTiterGlo reagent  was added to assay plates and incubated at room temperature for one hour (as opposed to ten minutes at SBP).MeWo and A-375 cell lines were obtained directly from American Type Culture Collection, all SK- cell lines were received directly from Memorial Sloan Kettering Cancer Center, TGen generated all UACC- cell lines, and all were of low passage number. Cells were maintained according to the manufacturer's or collaborator's instructions: A-375 cells are grown using DMEM medium, MeWo and Sk-Mel-2 cells are grown using EMEM medium, and UACC-0257 cells are grown using RPMI1640 medium. All media have 10% FBS and 1%AA added to final growth media. All cell lines were banked at low passages in multiple aliquots as liquid nitrogen stocks to reduce risk of phenotypic drift. All cells were cultured for less than three months before reinitiating culture from the frozen stock. All cells were routinely inspected for identity by morphology and growth curve analysis and validated to be mycoplasma free. All cell lines were free of contaminants.norm between the data obtained from each experimental pair setting at each dose  and the correlation coefficient across all available doses . Since the Spearman correlation is robust to outliers, is non-parametric, and does not necessarily assume linear relationships, we chose use it as implemented in base stats package [We assessed the variability of the HTS data by first examining the correlations between measures obtained at matching doses using the two SBP screens  and then across the SBP and TGen screens . We calculated the Spearman correlation of the CV package  in R. We package  for RFor a more comprehensive assessment of the assays’ variability and reliability on the four common cell lines across institutes, we used flexible linear models and ANOVA , cell lines (n = 4), and drugs (n = 6). All analyses were performed in R and all figures were generated using the graphics11 package in R.Because we were unable to access the raw experimental data for the Cancer Cell Line Encyclopedia (CCLE) data, we explored the consistency of the ICn Cancer  (GDSC) d


The isolation of clusters of circulating tumor cells (CTCs) from cancer patients has recently challenged the accepted view that the initiation of secondary tumors during metastasis involves the dissemination of individual cancer cells. As such clusters appear to be more aggressive than single tumor cells, CTC clusters are now considered a main player in the metastatic process, and many studies are exploring their diagnostic, prognostic, and clinical significance. However, several technical challenges limit advances in this area. Here, we suggest the use of established cancer cell lines that grow as cell clusters in suspension as a complementary approach that can help in understanding the biology of CTC clusters and their clinical significance. We argue that the many similarities between these “surrogate” clusters and the CTC clusters isolated from patients  make these cell lines ideal systems for the development of strategies aimed at preventing or slowing down the metastatic process by targeting CTC clusters. Cells iex vivo culturing of single CTCs is now possible [e.g., Ref.  as examples and proof-of-principle. NCI-H187 is a small cell lung cancer line  that grows as multicellular aggregates in suspension. NCI-H2122 is a non-small cell lung cancer line  that shows a mixture of both adherent cells and grape-like cell clusters in suspension. Under typical growth conditions, the two cell lines form clusters that are similar in size and conformation to CTC clusters isolated from patients with various cancer types—including lung, breast, and prostate cancer ., 5.4, 5)s Figure . FurtherAdditionally, such cell lines can be used to investigate the mechanisms that confer increased chemo- and radio-resistance upon CTC clusters . Drug-resistance can also be selected for in these lines, and new therapeutic approaches that can decrease the survival of resistant CTC clusters or interfere with their potential to establish new metastases can be tested. Cell lines with various genetic backgrounds and mutation panels are available, which can expand the range of studies addressing the effect of general or specific/targeted drugs on CTC clusters.Although the surrogate CTC clusters cannot provide insights into the first phase of the metastatic process , they can facilitate our understanding of the “colonization” phase—a process that has been identified as requiring additional research . For insin vivo CTC clusters and facilitate the development of strategies aimed at preventing or slowing down metastasis by targeting CTC clusters. We do recognize that there are limitations associated with the use of these “surrogate” CTC clusters, including the absence of non-cancer cells, the presence of which is thought to influence the properties and responses to therapy of real CTC clusters. However, we believe that the accessibility and affordability of such cell lines for both in vitro and in vivo studies and the potential for standardizing and replicating such studies  are features that can complement the more limited studies can be performed currently with isolated primary CTC clusters.In this perspective, we suggest that established or experimentally evolved cell lines that grow as cell clusters in suspension can improve our understanding of the biology of real AM, BC, and AN designed the study, interpreted the data, and wrote the manuscript. AM and BC carried out the experiments.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.


Intestinal microbiota disruption is associated with acute gastrointestinal (GI) Graft-versus-Host Disease (GvHD) and poor outcome after allogeneic stem cell transplantation (ASCT). Here, in a retrospective analysis of 200 patients undergoing ASCT at the Regensburg University Medical Center, we assessed the relative expression of Paneth cell antimicrobial peptides (AMPs), Human Defensins (HD) 5 and 6 and regenerating islet-derived 3α (Reg3α), in 292 human intestinal biopsies as well as Reg3α serum levels in relation to acute GI GvHD. In the absence of GI GvHD, the relative expression of Paneth cell AMPs was significantly higher in the small intestine (duodenum to ileum) than in the stomach and large intestine (cecum to rectum) for Reg3α (p≤0.001), HD5 (p≤0.002) and HD6 (p≤0.02). Acute stage 2–4 GI GvHD was associated with reduced expression of AMPs in the small intestine (p≤0.01) in comparison to stage 0–1 disease, accompanied by a decrease in Paneth cell count in case of severe acute GI GvHD (p<0.001). The opposite held true for the large intestine as we found stage 2–4 GI GvHD correlated with significantly higher expression of HD5, HD6, and Reg3α compared to mild or no acute GI GvHD (p≤0.002). Severe GI GvHD in both the lower and the upper GI tract also correlated with higher serum concentrations of Reg3α (p = 0.002). As indirect markers of intestinal microbiome diversity low levels of urinary 3-indoxyl sulfate levels were associated with severe stages of acute GI GvHD compared to mild stage or no acute GI GvHD (p = 0.05). In conclusion, acute GI GvHD correlates with intestinal expression of HD5, HD6 and Reg3α as well as Reg3α serum levels and is associated with intestinal dysbiosis. Clostridiales, seem to be crucial for the maintenance of immunological homeostasis and intestinal epithelial integrity and for providing anti-inflammatory effects as demonstrated in both mouse models [The gut microbiota play a central role in the outcome of allogeneic stem cell transplantation (ASCT). , 2 Durine models , 6 and he models , 7Paneth cells play an important role in the maintenance of a balanced gut microbiota composition and contribute to intestinal homeostasis and innate immunity.  Located In this retrospective study of patients undergoing ASCT, we analyzed relative gene expression of Paneth cell AMPs HD5, HD6 and Reg3α in 292 intestinal biopsies and determined serum levels of Reg3α in relation to acute GvHD of the GI tract.A total of 200 adult patients undergoing ASCT at the University Hospital Regensburg were included in our retrospective analysis. Inclusion criteria were hemato-oncologic disease requiring ASCT with an age above 18 years and receiving non-T cell depleted grafts. The Ethics Committee of the University Medical Center of Regensburg approved the study (02/220) and all patients participated after receipt of written informed consent. None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. Intestinal biopsies were obtained in patients with clinical suspicion of acute GI GvHD by endoscopy. In case of a second episode of an acute GI GvHD a re-endoscopy was performed including a corresponding biopsy harvesting. Beside specimens for routine microbiological and histological workup, an additional biopsy of a macroscopic suspicious mucosa  was obtained for scientific investigation. In addition, the protocol allowed inclusion of screening biopsies from asymptomatic patients early after ASCT. 72% of all biopsies were taken within the first 180 days post-transplant with a mean time of 55 days after ASCT since classic acute GI GvHD and late acute GI GvHD occur usually within the first 180 days after ASCT.  28% of tAll biopsies were stored at -20°C until analysis. For PCR analysis of HD5 and 6 and Reg3α, biopsies were classified with regard to the distribution of Paneth cells and the location of biopsy harvesting into stomach, small intestine comprising duodenum, jejunum and ileum and large intestine including biopsies from the colon and rectum. Paneth cell counts were evaluated in histological sections of intestinal biopsies. To further investigate the association between Reg3α serum levels and acute GI GvHD we performed Reg3α serum measurements within seven days around the day of biopsy harvesting. Urinary 3-Indoxyl sulfate (3-IS) levels analyzed previously and obtained within 7 days of endoscopy for inpatients and 14 days for outpatients were used for assessment of microbiome diversity at the time of biopsy. Furthermore, 3-IS measurements within the first 4 weeks after ASCT were available for all samples with Paneth cell measurements. Patients’ characteristics are shown in GvHD in the upper and lower GI tract was staged according to the Glucksberg grading system.  In case Quantitative real-time PCR (qPCR) was performed on a total of 292 intestinal biopsies for HD5, HD6 and Reg3α as described earlier.(16) RNAs were stored at -80 degrees to avoid long term degradation. Following RNA extraction, the quality of RNA was determined by bio-analyzer and RIN score was between 7 and 9.5’-AGA-CAA-CCA-GGA-CCT-TGC-TAT-CTC, reverse: 5’-GGT-TCG-GCA-ATA-GCA-GGT-GG; DEFA6, forward: 5’-GAC-CAG-GAC-TTT-GCC-GTC-TC, reverse: 5’-CAA-GTG-AAA-GCC-CTT-GTT-GAG-CC; REG3A, forward: 5’-ATC-CGC-TGT-CCC-AAA-GGC-TC, reverse: 5’-AGC-ACA-GAC-ACC-AGG-TTT-CCA-G; 18S, forward: 5’-ACC-GAT-TGG-ATG-GTT-TAG-TGA-G, reverse: 5’-CCT-ACG-GAA-ACC-TTG-TTA-CGA-C. [18S ribosomal RNA was used as a reference gene. Gene of interest was normalized to 18S rRNA thus relative expression was evaluated. Following gene-specific primers were used: DEFA5, forward: A-CGA-C. In 126 patients giving 175 biopsies Reg3alpha serum levels were analyzed within seven days around day of biopsy harvesting in duplicate by using indirect ELISA as previously described.  Mean wasIn a total of 63 patients 3-IS levels were analyzed at the time point of biopsy harvesting as recently described. 2).The number of Paneth cells was determined with a Zeiss Axioskop 40 microscope in hematoxylin and eosin (HE) stained sections of intestinal biopsies of the duodenum (n = 51). Additionally the number of Paneth cells was assessed in a randomly chosen cohort of 22 patients with colon biopsies (n = 34). Paneth cells were counted in at least 3 high-power fields (hpf) in the area of each biopsy showing the largest number of Paneth cells. The counts from each hpf were then averaged to give the number of Paneth cells per hpf. (13) An hpf was defined as the visible area in a 400 x magnification (0.307 mmAll slides were reviewed by two pathologists (one fellow and one attending) independently, not blinded to clinical data. In case of disagreement, the attending overruled the fellow’s results.Continuous data are presented descriptively as median (range). Group comparisons were performed by two-sided Mann-Whitney-U-Tests or correspondingly Kruskal-Wallis tests due to non-normal data distribution. Statistical analyses were performed using IBM SPSS Statistics 22 .-5 to 5.5x101) vs. 2.7x10-3  and 1.2x10-3  for HD5 and 5.1 (1.3x10-4 to 8.6x101) vs. 9.4x10-3  and 9.5x10-3  for HD6. Interestingly, we found also significantly higher Reg3α expression in the small intestine  than in the stomach  or large intestine , we found significant differences in relative expression between stomach, small intestine and large intestine according to the physiological distribution of Paneth cells in the GI tract. The relative expression of HD5 and HD6 was higher in histologically normal biopsies (n = 134) of the small intestine than the stomach and large intestine with 7.3 (8.7x10-5 to 8.0x101) to 3.2x10-2 , for HD6 from 5.1 (1.3x10-4 to 2.2x102) to 6.0x10-2  and for Reg3α from 1.1x101 (5.1x10-4 to 3.3x102) to 6.6x10-2 , respectively. Reduced expression of AMPs was accompanied by a decrease in Paneth cell count from 18.6 (0–79.0) to 2.8  in case of severe GI GvHD. In the large intestine, in contrast, stage 2–4 GI GvHD correlated with significantly higher expression of HD5, HD6, and Reg3α (-3 (0 to 2.1) to 3.4x10-2 , for HD6 from 8.5x10-3 (1.9x10-4 to 2.0) to 4.5x10-2  and for Reg3α from 3.3x10-3 (3.1x10-5 to 2.3x101) to 4.2x10-2 , respectively. A metaplasia of Paneth cells in the large bowel was not found in the presence of severe acute GI GvHD as we observed Paneth cells in 12 of 34 colon biopsies. Accordingly, mean Paneth cell count in the large intestine was measured with 0.3 (0–37.4) in patients with no/mild GI GvHD and with 0.3  in patients with GI GvHD stage 2–4, respectively. Severe GI GvHD in both the lower and the upper GI tract also correlated with higher serum concentrations of Reg3α  as compared to mild GI GvHD . Due to the low number of stomach biopsies an analysis of GvHD changes in AMP secretion was not possible for this subgroup of patients.Next, we investigated the impact of acute GI GvHD on the relative expression of Paneth cell AMPs in acute GI GvHD. Severe acute GI GvHD (stage 2–4) was associated with a significant decrease in AMPs in the small intestine compared to mild stage or no GI GvHD (stage 0–1) . We founnd Reg3α , as we oAs indirect markers of intestinal microbiome diversity, urinary 3-IS levels were measured within 14 days of biopsy harvesting. Severe stages of acute GI GvHD were associated with lower levels of 3-IS with 0.001 μmol/mmol creatinine (0 to 0.3 μmol/mmol creatinine) compared to 0.06 μmol/mmol creatinine (0 to 99.0 μmol/mmol creatinine) in patients with mild stage or no acute GI GvHD (p = 0.05). The correlation of Paneth cell number and 3-IS showed a trend (data not shown). Instead, researching into early microbiome disruption within the first 4 weeks after ASCT, a higher number of Paneth cells with 25.0 (0–79.0) was found in patients with early high 3-IS levels (> median) compared to 13.0 (0–33.0) in patients with early low 3-IS levels (p = 0.01).The composition of intestinal microbiota seems to play a key role in the pathophysiology of acute GI GvHD and has been reported to influence the outcome of ASCT recipients. , 2 PanetAMPs are small cationic peptide antibiotics with bactericidal activity against pathogenic bacteria, fungi and several viruses.  However,Several genetic defects in Paneth cells have been reported in patients with Crohn´s disease resulting in a weakened mucosal antimicrobial defense and alterations of the intestinal microbiota. These findings suggest a key role of these cells in the pathophysiology of IBD. Interestingly, NOD2/CARD15 polymorphisms, which have also been described as risk factors of GvHD by our group, are among the most important genetic defects in IBD. –21 FurthIn 2011, Ferrara et al. described Reg3α as biomarker in the serum of patients with acute intestinal GvHD. The mucosal damage due to intestinal inflammation leads to microscopic breaches in the mucosal epithelial barrier permitting a transmission of Reg3α from the mucus into the systemic circulation. In the literature, acute GI GvHD was found to be associated with distinct disruptions of the intestinal microbiota diversity.  One mechA further hint, that intestinal microbiota composition strongly correlates with the regulation of AMPs was found by Li et al. in mice. After initiation of cecal dysbiosis by antibiotic treatment with ceftriaxone a significant overproduction of defensins in the ileum and colon could be observed. However, a recovery of intestinal microbiota by fecal microbiota transplantation led to quick normalization of defensin levels. The correlation of Paneth cell loss with GvHD but also early suppression of 3-IS levels raises the question concerning the causal relationship of the observed associations between GvHD and loss of AMPs. On one hand, Paneth cell loss by GvHD might induce loss of AMPs and subsequent suppression of commensal bacteria as indicated by 3-IS levels. The strong impact of early 3-IS levels however also raises the possibility that early loss of commensals  facilitates Paneth cell destruction (e.g. via loss of epithelial protection by IL22  and lossIn conclusion, our data suggest an association between acute GI GvHD, Paneth cells and their expression of AMPs HD5, HD6 and Reg3α in intestinal biopsies and a consecutive release of Reg3α in the blood stream. The low concentrations of 3-IS levels reflecting a disruption of the intestinal microbiota diversity provide an indication of the relevance of AMP shifts in patients undergoing ASCT.


Characterization data were consistent with a cis arrangement for the dppm ligands and a bidentate coordination through carboxylate oxygens of the quin and kynu anions. These complexes were not able to intercalate CT-DNA as shown by circular dichroism spectroscopy. On the other hand, bovine serum albumin (BSA) binding constants and thermodynamic parameters suggest spontaneous interactions with this protein by hydrogen bonds and van der Waals forces. Cytotoxicity assays were carried out on a panel of human cancer cell lines including HepG2, MCF-7, and MO59J and one normal cell line GM07492A. In general, the new ruthenium(II) complexes displayed a moderate to high cytotoxicity in all the assayed cell lines with IC50 ranging from 10.1 to 36 µM and were more cytotoxic than the precursor cis-[RuCl2(dppm)2]. The cis-[Ru(quin)(dppm)2]PF6 were two to three times more active than the reference metallodrug cisplatin in the MCF-7 and MO59J cell lines.The complexes In the current work quinoline carboxylates were chosen as coligands in order to generate cationic complexes and also to explore the possible different coordination modes of the ligands.Due to this background of promising biological results in complexes containing the cis-[Ru(quin)(dppm)2]PF6 (1) and cis-[Ru(kynu)(dppm)2]PF6 (2) are reported. The interaction of these complexes with CT-DNA and BSA was investigated by circular dichroism (CD) and fluorescence spectroscopies, respectively. Besides, preliminary in vitro tests of cytotoxic activities against a variety of human cell lines are presented and discussed.In this work the synthesis and characterization of two new derivatives with formula3·3H2O and the ligands 1,1-bis(diphenylphosphino)methane (dppm), quinaldic acid, and kynurenic acid were used as received from Aldrich. The cis-[RuCl2(dppm)2] precursor complex was prepared according to the literature method   was solubilized in 100 mL of methanol and the mixture was heated during 20 minutes. Quinaldic acid  or kynurenic acid , respectively, for synthesis of complexes 1 and 2, was solubilized in 10 mL of methanol and deprotonated with triethylamine  and this solution was added dropwise on the precursor solution. After this processes, the mixture was stirred and refluxed for 48 h. The final solution was concentrated to ca. 5 mL and an aqueous solution of NH4PF6  was added for the precipitation of a yellow solid. The solid was filtered off and washed with water (3 × 5 mL) and diethyl ether (3 × 5 mL) and dried under reduced pressure.The precursor60H50F6NO2P5Ru: exptl  C, 60.97 (60.71); H, 4.93 (4.63); N, 1.10 (1.18). 1H NMR (CDCl3): δ 4.11, 4.73 , 6.26 , 6.99–7.86 , 7.91 , 8.22 , 8.31  ppm. 31P{1H} NMR (161.73 MHz - CDCl3): δ 9.70 ; –12.2 ; –144.7 . UV-vis : λ/nm (ε/M–1 cm–1) 256sh (4.33 × 104), 325 (8.00 × 103). Molar conductivity  is the quencher concentration, and Ksv is the Stern-Volmer quenching constant, which can be written asKq is the biomolecular quenching rate constant and τo is the average lifetime of the fluorophore in the absence of quencher (6.2 × 10−9 s) [Ksv by linear regression of a plot of F0/F versus [Q].Complexes were dissolved in sterile DMSO. For fluorescence measurements, the BSA concentration was kept constant in all samples, while the complex concentration was increased from 3.125 to 150 Kb) and number of complexes bound to BSA (n) were determined by plotting the double log graph of the fluorescence data using The binding constant , and R is the gas constant, from which the ΔH and ΔS of the reaction can be determined from the linear relationship between lnKb and the reciprocal absolute temperature. Furthermore, the change in free energy (ΔG) was calculated from the following equation:The thermodynamic parameters were calculated from the van't Hoff equation:2) containing HAM-F10 plus DMEM  or only DMEM depending on the cell line, supplemented with 10% fetal bovine serum (Nutricell) and 2.38 mg·mL–1 Hepes (Sigma-Aldrich) at 37°C in a humidified 5% CO2 atmosphere. Antibiotics  were added to the medium to prevent bacterial growth.Cells from the 4th through to the 12th passage were used. The different cell lines were maintained as monolayers in plastic culture flasks  supplemented with 10% fetal bovine serum containing concentrations of complexes ranging from 12.5 to 1600 µg·mL−1. Negative (no treatment), solvent (0.02% DMSO), and positive (25% DMSO) controls were included. Positive controls comprising cisplatin  were included. After incubation to 36.5°C for 24 h, the culture medium was removed. Cells were washed with 100 μL of PBS for removal of the treatments, after which they were exposed to 100 μL of culture medium HAM-F10 without phenol red. Then, 25 µL of XTT was added and incubated at 36.5°C for 17 h. The absorbance of the samples was determined using a multiplate reader (ELISA-Tecan-SW Magellan versus 5.03 STD 2P) at a wavelength of 450 nm and a reference length of 620 nm.The screening for cytotoxic activity of cell lines was assessed using the Colorimetric Assay In Vitro Toxicology-XTT Kit (Roche Diagnostics). For the experiments, 1 × 1050 response parameter (50% cell growth inhibition) calculated with the GraphPad Prism program, plotting cell survival against the respective concentrations of the treatments. One-way ANOVA was used for the comparison of means (P < 0.05). The selectivity index was calculated by dividing the IC50 value of the isolated compounds on GM07492-A cells by the IC50 value determined for human cancer cells.Cytotoxicity was assessed using the IC cis-[RuCl2(dppm)2] resulted in the products cis-[Ru(quin)(dppm)2]PF6 (1) and cis-[Ru(kynu)(dppm)2]PF6 (2), The simple reaction of the quinoline carboxylic acids with ruthenium(II) diphosphine precursor complex 1-2 exhibited satisfactory microanalytical  data. The 1H NMR spectra showed two broad signals due to the CH2 group of the dppm ligand close to 4.10 and 4.70 ppm  that are observed at 0.64 and −25.3 ppm with JPP = 36 Hz  and cisplatin were evaluated for their capability of inhibiting tumor cell growth in vitro using three human cell lines, HepG2, MCF-7, and MO59J. One nontumorigenic cell line (GM07492A) was also assayed in the same conditions in order to verify the selectivity of these compounds. The resulting concentration-effect curves obtained with continuous exposure for 24 h are depicted in 50 values) is listed in Ruthenium compounds 1, followed by complex 2, and the less active is the precursor cis-[RuCl2(dppm)2].Overall sensitivities of the three tumor cell lines Figures  and of t 1 and 2 have showed, in general, a moderate cytotoxicity against all the human tumor cell lines assayed with IC50 values ranging from 10.1 to 36 µM, except for complex 2 in MCF-7 cells that showed a very low cytotoxicity as shown in  1 displayed higher activity than 2 in all the cell lines assayed with IC50 close to 10 µM. This nonselective activity of complex 1 was not observed for complex 2 that was almost inactive in MCF-7 cells. The selectivity index (SI) was very close to 1  for all the cell lines assayed indicating a lack of selectivity for both complexes. In the same experimental conditions the precursor complex cis-[RuCl2(dppm)2] was less active than the complexes 1 and 2 by factors ranging from 1.4 to 15.8. A similar increased in activity was also observed against the normal cell line GM07492A. This lack of selectivity was also observed for the cis-[RuCl2(dppm)2] for all the cell lines assayed and for cisplatin in the MCF-7 and MO59J cell lines. These data clearly indicate that exchanging two chlorido ligands by a bidentate quinoline carboxylate group turns the unity cis-[Ru(dppm)2] complex more cytotoxic, probably due to its higher solubility and disponibility in the culture medium. Interestingly, the presence of one OH-group in complex 2 led to a significant decrease in cytotoxic activity, indicating that this may be a way to modulate the cytotoxic potency of this type of complex.Complexes cis-[Ru(quin)(dppm)2]PF6 and cis-[Ru(kynu)(dppm)2]PF6 were synthesized and characterized by elemental analysis and spectroscopic methods. Characterization data are in agreement with a cis geometry and a chelated coordination, through the carboxylate group, for the quinaldate and kynurenate ligands. Utilizing circular dichroism spectroscopy showed that these complexes lack the ability to intercalate DNA. On the other hand BSA-binding constants and thermodynamic parameters suggest spontaneous interactions with this protein by hydrogen bonds and van der Waals forces. The in vitro cytotoxicity activity assays indicate a moderate to high cytotoxicity against a panel of human tumor cell lines; however, these complexes lacks selectivity. Interestingly, modulation of cytotoxic potency can probably be done by exchanging the substituents of the quinolone carboxylate ligand and will be explored in future work.In this investigation two new ruthenium(II) complexes containing dppm and the quinaldate and kynurenate anions with formula


The collective response of these pathways to component drugs was both cell type- and drug type specific. However, the entire spectrum of pathways triggered by the tri-drug regimen was similar in all four cancer cell lines, explaining its broad spectrum killing of BCa lines, which did not occur with its component drugs. Our findings here suggest that the FSC platform holdspromise for optimization of anti-cancer combination chemotherapy.Therapeutic outcomes of combination chemotherapy have not significantly advanced during the past decades. This has been attributed to the formidable challenges of optimizing drug combinations. Testing a matrix of all possible combinations of doses and agents in a single cell line is unfeasible due to the virtually infinite number of possibilities. We utilized the Feedback System Control (FSC) platform, a phenotype oriented approach to test 100 options among 15,625 possible combinations in four rounds of assaying to identify an optimal tri-drug combination in eight distinct chemoresistant bladder cancer cell lines. This combination killed between 82.86% and 99.52% of BCa cells, but only 47.47% of the immortalized benign bladder epithelial cells. Preclinical  Although there have been significant advances in our understanding of the molecular basis of cancer and several hundred-targeted therapeutics were introduced based on these discoveries, chemotherapeutic regimens that are the mainstay of cancer treatment remain largely unchanged47881115An obvious but prohibitive approach is the testing of all possible combinations of all drugs at all doses for the best regimen of the markedly improved therapeutic index. However, an effort of this kind exceeds the screening capacity of today’s biomedical research laboratories. Moreover, the extensive heterogeneity at the genetic, epigenetic, expressional, and phenotypic levels of cancer cells in patients necessitates testing a large number of cancer cell lines in order to represent disease diversity, which further amplifies the task.Bladder cancer (BCa) is the fourth most common type of tumors in males worldwide1932224242526in vivo growth of three BCa-cell-line-derived-tumor-xenografts in nude mice than did its mono- or bi-drug constituents. By determining the activities of nine cancer-associated pathways in four bladder cancer cell lines and one immortalized normal cell line, we showed that the collective response of these pathways to the single component drug of the tri-drug combination is both drug type and cell line-specific. However, the entire spectrum of pathways activated by the tri-drug regimen was similar in all four cancer cell lines. This explains why the tri-drug combination has a broad anti-cancer effect but its component drugs fail. The broad applicability of FSC for the optimal drug combinations to a wide range of disease indications is fully anticipated.In this study, we used this platform to quickly identify an effective tri-drug combination that is capable of killing seven TCC cell lines and one squamous cancer cell line that better represent the clinical spectrum of bladder cancer than any studies using a single or fewer cell lines. In contrast, this regimen possesses a significantly lower killing capability on immortalized benign epithelial cells, an indication that its broad anti-cancer cytotoxicity. It also more effectively suppressed the https://www.nccn.org/store/login/login.aspx? ReturnURL =  http://www.nccn.org/professionals/physician_gls/pdf/bladder.pdf):Pirarubicin (Pi), Paclitaxel (Pa), Epirubicin Hydrochloride (EH), Cisplatin (Ci), Gemcitabine (Ge), and Mitomycin (Mi) in eight human bladder cancer cell lines  and an immortalized benign cell line (SV-HUC-1) (We used the MTT (Test by thiazolyl blue tetrazolium blue) based assay to determine the dose required for 50% of cells to be killed (IC50) by following six anti-bladder cancer drugs (V-HUC-1) . The relV-HUC-1) , to descV-HUC-1)  was calcV-HUC-1)  illustraV-HUC-1) , to SCaBV-HUC-1) . TherefoThe total number of possible combinations for six drugs at five doses is 15,625 for one cell line and 125,000 for all eight BCa cell lines. This represents a prohibitive scale of workload if all potential combinations are examined. To experimentally identify an effective tri-drug combination in this study, the DE algorithm version of the FSC platform was implemented   of the 20six-drug combinations tested in the 2nd round of iteration were effective (Table S2). To reduce the number of drugs in combinations from six to four or five, a zero dose option was introduced in the 3rdand the 4th round of testing for four or five drug combinations. The number of the effective combinations was raised from the 2nd round  (Table S2) to the 3rd round  (Table S3), and the 4th round  of testing  different drugs , the system output, y, can be expressed by a 2nd order polynomial regression model fits the experimental results of Biu87, 5637, UM-UC-3, and H-bc which were plotted for comparison , or the second highest doses (Ci = 300 and Mi = 200 ng/mL) alone or with one of the third drugs at one of three doses  impacted by conventional chemotherapeutic agents remain enigmatic, despite years of research effort. To provide the signaling pathway based mechanisms for BCa chemoresistance, we used a Signal Pathway Finder reporter system (Qiagen)  to deterWe then determined the correlation coefficient between the overall activity of these nine pathways and the chemoresistant state of these five cell lines. The pathway activity was digitized as follows: (1) an active state: the signaling pathway with an activity higher by 0.5 fold than in 5637; (−1) an inactive state: the signaling pathway with an activity lower by 0.5 fold than 5637; and (0) an inert state: the signaling pathway with an activity being neither of the above. The overall state of activity of these pathways in each cell line were summarized  and the The correlation coefficients of the overall nine pathway activity between 5637 and four chemoresistant cell lines were negative , and indicating the distinct state of 5637 from the other four cell lines, these figures in each pair were close to 0.99 . It was in vivo anticancer activity of the EH/Ci/Mi regimen with its mono-and bi-drug counterparts on the 5637, UM-UC-3, or Biu87 cells derived tumor xenografts in nude mice. Since there is no established conversion rule for the drug dose in cell culture to in nude mice, we used 75 μg per mouse for the mono-drug treatments and 25 μg per drug per mouse for both bi-drug and tri-drug combinations, based on the maximum tolerated drug dose recommended for these drugs313234We then compared the Comprehensive cancer genomic studies in the last few years have repeatedly demonstrated that genetic heterogeneity in cancer cells differentiates not only in tumors from different individuals, but also different lesions, different part in a single lesion, or different cancer cells in a cancer patient37The current anti-bladder cancer combination chemotherapies are chiefly cisplatin-based: CMV , M-VAC 41424445464749505153To understand why the EH/Ci/Mi regimen is capable of killing a broad spectrum of BCa cells but each component drug at a three fold higher dose failed, we analyzed the basal and drug-triggered activities of nine cancer associated signaling pathways in four bladder cancer cell lines  and an immortalized non-transformed bladder epithelial cell line (SV-HUC-1) (Table. S4): DNA damage response, Hypoxia, ER Stress, Heat Shock, Wnt, Notch, Cell cycling, Myc/Max, and MAPK/ERK pathways  combination therapy for cancer.Each experiment was performed three times. All animals were adult BALB/C male nude mice 8–12 weeks of age and experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the animal research committee of Harbin Medical University, China.http://www.cancer.gov/dictionary) was provided by the First Affiliated Hospital of Harbin Medical University: Pirarubicin , Paclitaxel , Adriamycin , Epirubicin Hydrochloride , Hydroxycamptothecin , Cisplatin , Gemcitabine , and Mitomycin . Drug induced cell death was determined by a thiazolyl blue tetrazolium blue  based cell proliferation assay in the cultured cells following treatment by various concentrations of drugs for 72 hours, as previously described50 as a reference. The overall chemoresistance of each cell line was described as the “chemoresistance index”, the numerator of the total relative IC50 to all the drugs over the number of drugs to rank the overall resistance state of each cell line to these six chemotherapeutics collectively.Cell lines (Table S1) used in this study were one normal uroepithelium cell line, SV-HUC-158“Differential Evolution” algorithm guided experimental optimization (DE) consists for three iterative operations: generating drug combinations for experimental testing, acquiring the efficacy of the generated drug combinations from biological assays, and recalculating further improved drug combinations using a feedback search algorithm. We first tested twenty drug combinations at randomized doses. These drug combinations were generated from a random integer matrix generator, coded in MATLABTM language. These twenty drug combinations were then experimentally tested for cancer cell cytotoxicity. The experimental data were then subjected to calculation with a DE algorithm for new drug combinations based on three major mathematical calculations: “Mutation”, “Crossover” and “Selection”. The equations for each step can be found in the main text. The equations for each step are coded in MATLABTM language.Statistical analysis of experimentally measured cell survival rates was done for each individual cell line. The readouts (ACS) from 100 tested options for each cell line were pooled together and a polynomial regression model was built. The polynomial regression modeling is commonly used to mathematically describe the relationship between Y’s  and X’s  using the R language environment. The polynomial regression model was computed by function “g = lm{stats}” and simplified by function “g = step(stats)” in R programming.Renilla luciferase construct. Transcriptional response element (TRE) sequence response elements to each specific transcriptional factor triggered by the different signaling pathways are positioned before the TATA box of the firefly luciferase reporter gene. The extent of activity of the reporter reflects the level of activity of the particular signaling pathway. The CMV controlled Renilla luciferase expression vector is co-transfected into the cell as an internal control. A basic reporter construct that lacks the transcriptional factor binding sequence and could not be induced by any transcription factors is used as a negative control. Cells transfected with the CMV directed firefly luciferase reporter gene serve as a positive control. Briefly, cells seeded in 6-well plates were transfected with Cignal finder pathway arrays according to the manufacturer’s instructions (Qiagen)Renilla luciferase) was performed using a Promega GloMax 20/20 illuminometer. The firefly luciferase activities were normalized over the Renilla luciferase activities after background activity determined by luciferase activities of negative controls was subtracted. A triple test of cell survival under these drug doses was also performed by MTT assay following 18 h incubation at 37 ºC.The pathway analysis was performed with the Qiagen’s Cignal Finder Pathway Reporter Array systems according to the manufacturer’s instructions. The pathway-focused dual-luciferase reporter construct is a mixture of an inducible transcription factor responsive firefly luciferase reporter and constitutively expressing The levels of pathway activities were digitized as follows: activated (1): if pathway activity was higher than 1.5-fold over the activity in 5637 cells  or than 6–7th/100 μL) embedded in BD Matrigel™ Matrix2L × 0.5 (where W and L represent the largest and second largest tumor diameters (mm)) and then plotted. The tumor mass were weighed, and fixed/sliced for immunohistochemistry after the animals were sacrificed.Cells  against both Ki67 and CD34. Pictures were taken by a LEICA DM 4000B microscope and the relative level of each protein was calculated using LEICA software. The relative staining per antibody (in percentage) in the mock treated over chemotherapeutic treated tumors was calculated and plotted.in vivo study involved four groups with three mice per group. Both mean ± S.D of tumor volume and weight were calculated. Student’s t-test was used for statistical comparisons. P values less than 0.05 were considered statistically significant.Two sets of tumor xenograft/nude mice studies were carried out. The How to cite this article: Liu, Q. et al. Preclinical optimization of a broad-spectrum anti-bladder cancer tri-drug regimen via the Feedback System Control (FSC) platform. Sci. Rep.5, 11464; doi: 10.1038/srep11464 (2015).