Brain tumor glioblastoma has no clear molecular signature and there is no effective therapy. In rodents, the intracellular distribution of the cyclic AMP (cAMP)-dependent protein kinase  R2Alpha subunit was previously shown to differentiate tumor cells from healthy brain cells. Now, we aim to validate this observation in human tumors. The distribution of regulatory (R1 and R2) and catalytic subunits of PKA was examined via immunohistochemistry and Western blot in primary cell cultures and biopsies from 11 glioblastoma patients. Data were compared with information obtained from 17 other different tumor samples. The R1 subunit was clearly detectable only in some samples. The catalytic subunit was variably distributed in the different tumors. Similar to rodent tumors, all human glioblastoma specimens showed perinuclear R2 distribution in the Golgi area, while it was undetectable outside the tumor. To test the effect of targeting PKA as a therapeutic strategy, the intracellular cyclic AMP concentration was modulated with different agents in four human glioblastoma cell lines. A significant increase in cell death was detected after increasing cAMP levels or modulating PKA activity. These data raise the possibility of targeting the PKA intracellular pathway for the development of diagnostic and/or therapeutic tools for human glioblastoma. Glial tumors of the brain are classified into four grades according to World Health Organization staging. Glioblastoma multiforme (GBM) is the most malignant (grade IV) glial tumor, with a dismal prognosis, despite recent therapeutic advances. GBM is highly invasive and recurs in nearly 90% of cases at the original site . MultiplPKA are tetramers of two regulatory and two catalytic subunits, present in every cell where they regulate several processes . When thdf = 1, p < 0.001 (Inspection of data from the The Cancer Genome Atlas (TCGA) database was performed on the expression signals for the regulatory PKA subunits in 540 cases of GBM, compared to non-tumor brain samples from the Genotype Tissue Expression (GTEX) study . R2A app < 0.001 a). These < 0.001 . ApparenThe presence of the catalytic and regulatory subunits of PKA was examined by Western blot in four human GBM cell lines, fractionated into soluble and membrane-bound fractions. All the four cell lines were positive for nestin b, NES. BThe insoluble and soluble fractions of human glioblastoma biopsies were compared to the corresponding fractions of the primary culture derived from the same biopsy, showing the same pattern of reactivity for both R2 and CD133 c, case 6To establish whether the cAMP-activated intracellular signaling pathway could be a target for glioblastoma pharmacological treatment, we tested a panel of cAMP/PKA-affecting agents, and examined their effect on survival of four human GBM cell lines. Interference with the cAMP–PKA pathway induced cell death . A signiThe in vitro data showed that PKA levels could vary in tumor cells, and that PKA activity could influence cell survival. The next step was to examine the complex brain tumor tissue for the distribution of PKA subunits. Immunofluorescence on the GBM specimens showed that R1 was present in isolated clusters throughout the tissue, in variable grades: under the present conditions, some GBM specimens were not labeled at all, while others presented clusters of R1 in some areas of the tumors . A dotteAt the cellular level, PKA R2 subunit distribution was reminiscent of that observed in cell cultures, and co-localized with a Golgi marker u. The saTo date, the treatment options for malignant gliomas are disappointing. Interference with intracellular signaling pathways that modulate cell proliferation may be a viable target. GBM presents neither clear molecular signatures nor peculiar genetic alterations , and thePRKAR2B gene resides, and alterations in chromosome 17, which hosts the PRKAR1A gene. In this way, the balance of PKA R1 to R2 subunits is altered [The anomalies found in GBM are diverse and numerous. Among the most frequent are the gain of chromosome 7, in which the  altered . This baInteractions among different intracellular pathways ultimately determine the cellular response. Hence, even minor changes in the organization of a single pathway may have large and unexpected consequences on cell function. Moreover, activation of the signaling pathway, that controls cellular functions critical to maintaining cancer characteristics, may be reversed by changing intracellular conditions. For example, an increase in cAMP concentration may shift the activation pattern from R1 (high affinity) to R2 (low affinity) subunits, in turn affecting their downstream effectors . It has In four human glioma cell lines, R2 and the catalytic subunit may be found in soluble and particulate cell fractions. In contrast to what is seen in normal brain primary cultures, both the glioma cell line and GBM primary culture present a diffuse R1 cytoplasmic labeling and a striking hotspot of R2 co-localized with the Golgi marker, similar to what was observed in rat and mouse GBM cell lines . TherefoImmunohistochemistry on GBM biopsies revealed that R1 is scarcely present (in isolated hotspots) in some specimens. Even the catalytic subunit is present in discrete clusters. The most striking feature of GBM samples is however the consistent R2 immunoreactivity in the tumor cells, located at the Golgi, an association that was previously observed in different cells . R2 distThese data suggest that R2 immunoreactivity may be exploited for diagnostic purposes in GBM samples. However, it should be considered that after irradiation and chemotherapy, the tissue was too autofluorescent to be examined with reliability. The amount of intracellular cAMP varies during cell cycle in malignant gliomas, being higher in G0/G1 and lower at mitosis: cAMP arrests the cell cycle and induces differentiation and apoptosis, possibly by altering the rate of PKA subunit degradation , in thisIEX-1 [Of note, the effects of cAMP on glioma cells are reversible, and a few cells are not sensitive to cAMP action . TherefoIEX-1 , and GFAIEX-1 ,40. GFAPConceivably, the cAMP system is not a suitable pharmacological target because of its universal distribution and its implication in almost every cellular process, in both normal and pathological cells. Therefore, a more selective targeting of GBM-specific features should be exploited. If generic PKA acting agents are not specific enough and may induce off-target unwanted effects, other strategies could involve the targeting of pathways that use PKA in GBM-specific processes. For example, it could be interesting to act on different pathways that use PKA as an effector. Various dopamine receptors have been implicated in GBM stem cell survival and proliferation . They acTherefore, the activation of PKA in glioma cells induces several processes, that ultimately lead to differentiation. If the presence of R2A is higher in GBM than in healthy brain tissue, then it would be desirable to reduce it, with the aim of sponsoring the expression of the other PKA subunits that are necessarily induced for healthy cell survival.PRKARIIA, PRKARIIB, PRKARIA and PRKARIB) in GBM samples (n = 540). Data were interrogated using the r2 platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi) to obtain Kaplan–Meier plots that were thresholded at the best expression level predictive of survival. Independent validation was obtained by interrogating the REMBRANDT database, which collects data from 178 GBM patients generated with Affymetrix® HG U133 v2.0plus using the BETASTASIS resource (http://www.betastasis.com/glioma/rembrandt/), thresholding at the median value to discriminate high and low expression. The TCGA database was also explored with the University of California Santa Cruz (UCSC) XENA browser (https://xenabrowser.net/), to compare the data from GBM patients to data obtained from normal brains in the GTEX study (n = 158), generated with Illumina TrueSeq RNA sequencing and Affymetrix® Human Gene 1.1 ST Expression Array V3. The analysis of survival was performed with chi-square comparing the two groups .The TCGA database was searched to obtain gene expression data for PKA regulatory subunit coding genes . Patients gave their written informed consent for taking the samples  and were recruited at the University of Padova Medical School. Twenty-eight patients were examined , unless otherwise stated. Reagents for cell cultures were from Gibco .PTEN and CDKN2A), U87 , and LN229 (mutated in P53) were obtained from ATCC . Human GLI36 glioblastoma cells were obtained from the University of California [2. Primary cell cultures were obtained from biopsies (see above) and maintained in DMEM supplemented with antibiotics and 10% fetal bovine serum [Human high-grade glioma cell lines A172  with protease inhibitor cocktail  and forced 40 times through a 27-gauge needle. They were then centrifuged for 15 min at 6000 rpm at room temperature. The supernatant was collected, and the pellet was washed again with the same volume of PBS and centrifuged as above twice. The second and third supernatants were discarded and the pellet resuspended in the same volume of PBS. The protein extraction from the 30 mg biopsy was performed as above in 40 volumes of PBS. Pellets and the first supernatant were run on a 12% polyacrilamide gel, then blotted on nitrocellulose membrane, blocked for 60 min with 1.5% bovine serum albumin, and incubated overnight with primary antibodies against: PKA catalytic subunit, PKA RIIA subunit, or PKA RIB subunit , CD133  or nestin , incubated for 2 h with horseradish peroxidase (HRP)-conjugated antirabbit secondary antibody  and revealed through chemiluminescence . Cells were grown in coverslips, washed in PBS, and frozen. After thawing, they were incubated for 5 min in 2% Triton X-100 in PBS at room temperature and fixed in 5% formalin and 1% Triton X-100 for one minute at 37 °C. Tissues were cut on a cryostat at 20 μm. Slides were air dried and post-fixed for one hour in 5% formalin at 18 °C, followed by 30 min in 2% Triton X-100 in PBS. Primary antibodies were incubated overnight. The following antibodies were used : RIIA (rThe present data suggest that the intracellular distribution of the different PKA isoforms is characteristic of each cell type. PKA distribution varies during physiological modifications and/or pathological transformations, and could be exploited as a diagnostic or therapeutic target in glioblastoma.


MET amplification but exhibited the epithelial-mesenchymal transition (EMT) phenotype. Overexpression of EMT inducers, Snail or Slug, in the parental lines promoted their resistance to gefitinib. The gefitinib-resistant cell lines regained their sensitivity to gefitinib and displayed reverse EMT phenotypes after long-term culture in gefitinib-free culture medium. Blockage of reverse EMT by stable expression of Snail or Slug prevented the regain of TKI sensitivity. In conclusion, reverse EMT is one of the major mechanisms for the regain of TKI sensitivity in TKI-resistant NSCLC cells, suggesting that the development of small molecules targeting the EMT process may prolong the efficacy of TKIs in NSCLC patients with EGFR mutations.Tyrosine kinase inhibitors (TKIs) are currently the first-line treatment for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. These patients receive platinum-based chemotherapy as the second-line treatment after they develop resistance to TKIs. Many patients regain sensitivity to the TKIs used in the first-line treatment after the failure of chemotherapy. However, the molecular mechanism for the regain of TKI sensitivity is largely unknown. In this study, we established gefitinib-resistant PC9 and HCC827 cell lines, which did not harbor the EGFR T790M mutation and  Approximately 50% of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion and L858R point mutation, receive tyrosine-kinase inhibitors (TKIs) as their first-line treatment. Despite the initial response to TKI therapy, the tumor eventually recurs due to acquired drug resistance to the TKIs. In 50% of these patients, the resistance against TKIs can be explained by secondary EGFR mutations, mainly T790M point mutation. A third-generation TKI, osimertinib, targeting the EGFR T790M mutation has been recently developed and prolongs disease-free survival of NSCLC patients [human epidermal growth factor receptor 2 (HER2) amplification, and activation of secondary pathways, such as MET-mediated pathway [Although osimertinib is very effective in lung cancer patients with the secondary EGFR mutation, the other 50% of patients who do not develop the T790M mutation receive platinum-based chemotherapy as their second-line treatment, and the prognosis of these patients is poor . Interes pathway . HoweverEpithelial-to-mesenchymal transition (EMT) is a reversible process that controls the phenotypic changes due to cell mobility , metabolEMT has long been linked to resistance of lung cancer cells to chemotherapy , 19 and 2 at 37°C. For long-term culture, all the cell lines were tested for mycoplasma every month. The gefitinib-resistant sublines were established by stepwise escalation of gefitinib concentrations from 0.5 μM to 10 μM. Gefitinib was purchased from LC Laboratories . The culture medium was replaced every 3 days. After 6 months, the resistant cells were regularly maintained in culture medium containing 10 μM gefitinib. For gefitinib-withdrawal experiments, the doubling time for the PC9-WGR and HCC827-WGR cells was determined, approximately 36.8 hours and 55.7 hours, respectively. During the gefitinib withdrawal, the PC9-WGR and HCC827-WGR cells were passaged every 3 days, and the cells were seeded at 30–40% confluency.The human non-small cell lung cancer cell lines PC9 and HCC827, carrying EGFR exon19del E746-A750, were purchased from American Type Culture Collection. Both cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. The cell lines were kept in a humidified incubator containing 5% CO3 cells/well were seeded in 96-well microtiter plates. Different concentrations of gefitinib were added 24 hours after seeding. After 72 hours of incubation, CellTiter® 96 non-radioactive cell proliferation assay  was performed according to the manufacturer’s instructions, and the optical density was determined at 560 nm by using a microtiter plate reader . The mean absorbance at each drug concentration was normalized to the control. The IC50 was defined as the drug concentration that caused a 50% reduction in relative absorbance.To determine the cytotoxic effect of gefitinib in sublines of PC9 and HCC827, cells at the density of 5×10Slug , Snail  and Twist1   were cloned into the pLex-MCS lentiviral vector. The shRNA-containing lentiviral vectors were provided by the National RNAi Core Facility, Academia Sinica, Taiwan. The identifier (ID) numbers of the two shRNA clones used for E-cadherin knockdown are as follows: TRCN0000130433 (shE-cad #1), TRCN0000131097 (shE-cad #2). The lentiviral particles for all expression plasmids and shRNAs were prepared by co-transfection with SPAX2 and pMD2G plasmids into HEK293T cells. Stable clones of individual infected cell lines were established by using puromycin (1–3 μg/mL).For generation of the gene expression lentiviral plasmids, synthesized DNA fragments of Total RNA isolation and reverse transcription were conducted using the method described previously . QuantitFor Western blot analysis, the cells were harvested and lysed in 1X RIPA buffer containing protease inhibitors and phosphatase inhibitors . Protein concentration was determined using a Bio-Rad DC protein assay kit . Total protein (20 μg) was loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. Specific antibodies against E-cadherin , N-cadherin , Vimentin , Snail , Slug , and Twist1  were applied to detect the targets. Monoclonal anti-Actin antibody  was used as the loading control. The following secondary antibodies were used: goat anti-rabbit or anti-mouse immunoglobulin G (IgG)-conjugated horseradish peroxidase . To detect the specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System .p<0.05 was considered significant. Statistical analyses were performed using SPSS  software.For the quantitative real-time PCR and cell viability assay, three independent experiments were conducted, and three technical replicates for each sample in every independent experiment were performed. For all Western blot analysis, three independent experiments (cell lysate from different passages of the cell cultures) were performed. The quantitative data are presented as the mean±SD. Two-sided Student’s T-test was performed. 50) of gefitinib for PC9-GR (24.0±7.2 μM) and HCC827-GR (25.7±3.2 μM) sublines were dramatically increased compared to their parental cell lines   . Unlike ctively) . The HCCctively) . By usinll lines . In parall lines . The occll lines .50 = 0.04±0.02 μM, 9.0±1.8 μM and 11.8±2.0 μM for the PC9-pLex, PC9-pLex-Slug and PC9-pLex-Snail cells, respectively)   (Slug has been identified as an EMT inducer and contributes to gefitinib resistance in NSCLC . In addictively) . In the ctively) . Similarctively) . Since tctively) . Howeverctively) .Clinical observations showed that lung cancer patients with EGFR mutations who developed TKI-resistance and received chemotherapy as the second-line treatment remained responsive to the original TKIs as the third-line treatment. To demonstrate this possibility in vitro, gefitinib was removed from the culture medium (PC9-WGR and HCC827-WGR) of the PC9-GR and HCC827-GR sublines, which were maintained in gefitinib-containing culture medium. The cell morphology, gefitinib response and EMT status of the parental, GR and WGR PC9 and HCC827 sublines were monitored every 10 passages. Bright-field microscopy images showed that the cell morphology of the PC9-WGR subline gradually reverted to the spindle-like shape of the parental PC9 cells after 30 passages . Accompa50 of gefitinib in the PC9-WGR subline, while the IC50 of gefitinib was significantly reduced in the vector-only control group. In parallel, the forced expression of Slug or Snail blocked the regain of gefitinib sensitivity in the HCC827-WGR subline. Together, these results demonstrated that the regain of gefitinib sensitivity after prolonged withdrawal of gefitinib was mediated by reverse EMT in the TKI-resistant lung cancer cells.To investigate whether reverse EMT is the cause of the regain of gefitinib sensitivity, Slug or Snail was ectopically expressed in the PC9-GR and PC9-WGR sublines. In the vector-only control group, reverse EMT occurred after prolonged withdrawal of gefitinib from the culture medium. Ectopic expression of Slug or Snail diminished the increase in E-cadherin and decrease in N-cadherin and Vimentin in the PC9-WGR subline . SimilarSince the discovery of TKIs, targeted therapy has become the most effective way to manage lung cancer. Despite the initial response of patients to TKIs, most of the patients still experience tumor recurrence due to acquired resistance to TKI and receive chemotherapy as their second-line treatment . HoweverIn addition to findings in vitro, EMT has been linked to the poor clinical outcomes of lung cancer patients , 28, espMET amplification) does not alter the gefitinib-resistant phenotype of the cells [MET amplification regain the sensitivity to gefitinib [MET amplification, but displayed EMT phenotypes. Similar observation was also reported from other groups [Dose-escalation protocols have been widely used to establish TKI-resistant NSCLC cell lines with EGFR mutations. However, different experimental procedures between the protocols may lead to phenotypic or genotypic variations of the TKI resistant cells. For example, continuous exposure of PC9 cells to increasing concentrations of gefitinib results in the frequent T790M mutation, but intermittent treatment did not . Importahe cells , 33, butefitinib , which iefitinib . Howeverr groups , 34. Ourr groups . Due to Unlike Snail and Slug, the overexpression of Twist1 did not induce EMT under our experimental conditions, although these three transcription factors inhibit the expression of E-cadherin , 36. It Many environmental factors and intracellular proteins that can promote EMT have been identified . However


CCND2) is a member of the D-type cyclins, which plays a pivotal role in cell cycle regulation, differentiation and malignant transformation. However, its expression status and relative regulation mechanism remains unclear in renal cell cancer (RCC). In our study, the mRNA expression level of CCND2 is down-regulated in 22/23 paired RCC tissues (p<0.05). In addition, its protein expression level is also decreased in 43/43 RCC tumor tissues compared with its corresponding non-malignant tissues (p<0.001). We further detected that CCND2 was down-regulated or silenced in 6/7 RCC cell lines, but expressed in “normal” human proximal tubular (HK-2) cell line. Subsequently, MSP and BGS results showed that the methylation status in CCND2 promoter region is closely associated with its expression level in RCC cell lines. Treatment with 5-Aza with or without TSA restored CCND2 expression in several methylated RCC cell lines. Among the 102 RCC tumors, methylation of CCND2 was detected in 29/102 (28%) cases. Only 2/23 (8.7%) adjacent non-malignant tissues showed methylation. We then analyzed the correlation of clinical features and its promoter methylation. Collectively, our data suggested that loss of CCND2 expression is closely associated with the promoter aberrant methylation.Cyclin D2 ( RB), a critical negative regulator of G1/S transition [RB inactivates it and leads to the activation of transcription factors such as E2F suppressed by RB, which then increased the activation of transcription of genes involved in DNA synthesis and induced cells to enter S phase [In mammals, loss control of cell cycle is a distinctive feature of human carcinogenesis. The regulation of cell cycle mainly associated with the orderly change of phase-specific protein, such as cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors . Among tansition . The pho S phase . Owing t S phase ,6.CCND2, located at chromosome 12p13, is a member of the D-type cyclin family and mainly mediates the extracellular signaling environment with cell cycle progression [CCND2 was generally considered as a protooncogene in various tumors, overexpression of CCND2 has been reported in testicular germ cell tumor cell lines [CCND2 expression was observed in breast [CCND2 has also been reported to be the only one of D-type cyclins, being up-regulated in the conditions of growth arrest, ectopic expression of CCND2 blocked the progression of cell cycle [CCND2 might function as a tumor suppressor gene in a cancer-type dependent manner.Cyclin D2/gression . CCND2 wll lines , colon cll lines  and gastll lines ,10. Althll lines  and inhin breast ,13–15, ln breast , pancrean breast  and gastn breast . CCND2 hCCND2 has been reported in various cancers and the mechanism underlying CCND2 silencing in these mentioned cases is the aberrant promoter methylation [CCND2 promoter methylation was more frequently observed in high Gleason score tumors [CCND2 and the regulation mechanism underlying renal cell cancer has not been reported before.Aberrant promoter methylation of tumor suppressor gene is a common feature of renal cell cancer –21 and ahylation ,14–18. Ae tumors  and was e tumors .However,CCND2 in different type of tumors, we aimed to assess the expression of CCND2 and the status of promoter methylation in RCC cell lines, tumors and adjacent non-malignant tissues. Our results showed that CCND2 was down-regulated in RCC cell lines and primary RCC tissues. Moreover, the methylation status of CCND2 was more frequently observed in RCC tissues than adjacent non-malignant tissues. Demethylation treatment of RCC cell lines restored CCND2 expression, which suggested that epigenetic alteration might be the mainly regulated manner of CCND2 expression.Owing to the controversial role of The experimental methods and usage of clinical samples were approved by the Ethics Committee of the Peking University First Hospital. All these specimens were diagnosed by two urological pathological pathology physicians with patients’ written consent.2 (5%) incubator.The RCC cell lines 786-O, A498, CAKI-1, CAKI-2, OSRC, 769P, KOTO3 and HK-2  were originally obtained . These cell lines were routinely maintained in RPMI1640 or DMEM medium with 10% fetal bovine serum (FBS) , 1% penicillin G, and 1% streptomycin at 37°C in humidified COAll the RCC samples (fresh tissues and paraffine section tissues) were obtained from the Urology Department, Peking University First Hospital, Beijing, People’s Republic of China. All the RCC tissues and corresponding non-malignant tissues were collected from radical surgical resection without adjuvant therapy. All these specimens were diagnosed by two urological pathology physicians with patients’ written consent. A 2002 AJCC TNM stage and a Fuhrman nuclear grade were used for the classification of the tumor histopathology.For RNA extraction, fresh frozen tissues and cell lines were homogenized in TRI Reagent  and isolated as previously described . Total gCCND2 in RCC cell lines, reverse transcription PCR (RT-PCR) with GoTaq® Green Master Mix  and Real-time PCR with  were used according to the manufacturer’s protocol. Then we used Real-time PCR to examine CCND2 mRNA status in 23 paired RCC tumors and adjacent non-malignant tissues. The primers were: CCND2-F: CATGGAGCTGCTGTGCCACG; CCND2-R: CCGACCTACCTCCAGCATCC. The RT-PCR reaction condition was 35 cycles . Real-time PCR was done for 45 cycles . GAPDH primers were: GAPDH-F: TCCTGTGGCATCCACGAAACT; GAPDH-R: GAAGCATTTGCGGTGGACGAT, which was used as an internal control.To evaluate the mRNA expression of 6 cells/ml in six-well plate, after overnight culture, cells were treated with 10μM demethylation agent 5-Aza (5-aza-2’-deoxycytidine) for 96 hours, some RCC cell lines were further treated with 100 nmol/L histone deacetylase inhibitor TSA (trichostatin A) for additional 24 hours. After the treatment, the cells were collected for DNA and RNA extractions.Six RCC cells were primarily seeded at a density of 1x10CCND2 promoter to indentify the CCND2 promoter methylation status. The methylated and unmethylated primers of CCND2 were CCND2-m1: TACGTGTTAGGGTCGATCG; CCND2-m2: CGAAATATCTACGCTAAA-CG (276-bp product); CCND2-u1: GTTATGTTATGTTTGTTGTATG; CCND2-u2: TAAAATCCACCAACACAATCA (223-bp-product). The MSP was conducted in a 12.5μl reaction solution with GoTaq® Green Master Mix  for 35 cycles . PCR products were analysed in 2% agarose gels. To further confirm our MSP results, BGS was used to analyze the detailed methylation status. The BGS primers were CCND2-BGS1: GGAGGAAGGAGGTGAAGAA; CCND2-BGS2: CCCCTACATCTACTAACAAAC. 40 cycles/58°C annealing temperature of PCR were performed using GoTaq® Green Master Mix. The PCR products were connected into the pEASY-T5 zero vectors , then 5–9 colonies were randomly selected and sequenced.MSP and BGS were performed on the bisulfite-modified DNA samples using primer sets targeting the CpG-rich region in CCND2  in 1:250 dilution. All the photographs were taken randomly and measured using Image Pro Plus .Immunohistochemistry was carried out on forty-three paraffin-embedded RCC tissues and paired adjacent non-malignant tissues microarray with the primary antibody CCND2 vectors were bought from company . Then 786-O and OSRC cells were transfected with pLVX-IRES-ZsGreen1 and pLVX-IRES-ZsGreen1-CCND2 using Lip 3000 kit (Invitrogen) according to the manufacturer’s instruction.pLVX-IRES-ZsGreen1 and pLVX-IRES-ZsGreen1-CCND2 and vector were harvested and then plated in 96-well plate at a density of 2000 per well and incubated overnight at 37°C. The Cell Counting Kit-8  was used to analyze the cell proliferation. 10μL CCK-8 solution was added into each well with 100μL serum-free optional medium and incubated for 40 minutes. The absorbance was measured at 450nm. All the results were obtained from three independent experiments.Cells transfected with t test, Fisher’s exact test and Chi-square test were used. Statistical analyzed using the SAS version 9.0 software. A p-value was considered significant when less than 0.05.Data were presented as mean ± standard deviation (SD). Student’s http://www.proteinatlas.org/) had previously revealed that, CCND2 expression level was significantly lower in RCC tissues. To check the validity of these findings, we performed Real-time PCR to evaluate the mRNA status of CCND2 in 23 paired RCC tumors and adjacent non-malignant tissues. The expression of CCND2 was noted in 22/23 adjacent non-malignant tissues. In contrast, only 1/23 adjacent non-malignant tissue showed slightly decreased expression of CCND2 compared with RCC tumor tissue (p<0.05). Moreover, we used immunohistochemical staining to examine the CCND2 protein expression in 43 RCC tumors paired with adjacent non-malignnat tissues. We found that CCND2 was downregulated in RCC tissues inordinately. Interestingly, CCND2 protein expression was decreased in 43/43 RCC tumors compared with the corresponding adjacent non-malignant tissues . Thus, in both mRNA and protein status, specific loss of CCND2 was observed.Serial analysis of gene expression  cell line, RT-PCR was used with GAPDH as an internal control. As is shown in CCND2 was significantly down-regulated in 6/7 RCC cell lines and weakly decreased in CAKI-2 cell line comparing with HK-2 cell line. This is accordance with the results of RCC clinical samples. As is known to all, both genetic and epigenetic aberrant alteration influence the expression of genes. By analyzing the promoter region, we found two typical CpG islands in CCND2 promoter region (http://cpgislands.usc.edu/). To further assess the relationship between epigenetic regulation and CCND2 expression, methylation specific PCR (MSP) was performed to evaluate the methylation of CCND2 promoter. Hypermethylation at CpG-rich region with no mRNA expression of CCND2 was detected in 5/6 RCC cell lines , but was not detected in CAKI-2 and HK-2 with CCND2 mRNA expression  or treatment with histone deacetylase inhibitor trichostatin A (TSA) should result in the up-regulation of CCND2. Indeed, when six RCC cell lines were treated with 5-Aza with or without TSA, five out of six cell lines showed a differently increased expression of CCND2 , two with low CCND2 expression . 769P and 786-O showed higher methylation status than HK-2 cell line, which is consistent with the previously results of MSP. From this part we concluded that hypermethylation may lead to the CCND2 silencing in RCC cell lines. Meanwhile, BGS results were consistant with the MSP result before or after pharmacological demethylation treatment  was performed. The CpG-rich region of reatment . These rCCND2 promoter, MSP was used to examine the methylation status in 102 RCC tumors and 23 adjacent non-malignant tissues. As is shown in CCND2 was observed in 29/102 (28%) of RCC tumors, but only 2/23 (8.7%) in adjacent non-malignant tissues . CCND2 mRNA was also decreased in 6/7 RCC cell lines as the RT-PCR results showed. Furthermore, lack of protein expression was also observed in 43 paired RCC samples (p<0.001). After ectopic expression of CCND2, we found the ability of tumor growth was inhibited. These findings may indicate a tumor suppressive role of CCND2 in RCC, and its down-regulation may be related to tumorigenesis.As previously reported, overexpression of c factor . It was f tumors ,9. In rest, lung ,13–15 anst, lung . The differation , but it G1 phase . Thus, wCCND2 by aberrant promoter methylation has been reported in many kinds of tumors. In prostate cancer, the frequency of CCND2 methylation status was much higher in prostate cancer samples than in non-malignant prostate tissues (p<0.005). By analyzing the clinicopathological correlations, greater CCND2 methylation frequency was found to occur in high Gleason score group and in patients with higher mean PSA value. In addition, methylation of CCND2 correlates with prostate poor prognosis [CCND2 mRNA (18 of 24) and protein (10 of 13). In contrast, CCND2 was expressed in normal luminal and myoepithelial breast tissues. The silencing of CCND2 expression was associated with its promoter aberrant methylation by MSP. The methylation of CCND2 promoter was also detected in ductal carcinoma in situ, which suggested that loss of CCND2 is an early event in tumorigenesis and may associated with the evolution of breast cancer [CCND2 expression by promoter methylation is also observed in gastric cancer [CCND2 in renal cell cancer, we then test the methylation of CCND2 promoter as a possible cause that lead to the silence of this gene.Multiple mechanisms underlying inactivation of genes have been reported in various types of cancers, such as loss of heterozygosity, point mutations, homozygous deletions, and aberrant promoter methylation . Aberranrognosis . Likewist cancer . The absND2 mRNA  of 24 anCCND2 was significantly higher in RCC tissues than in adjacent non-malignant samples  of RCC patients. Many genes are known to be aberrantly methylated in RCC [CCND2 may be an useful part of the panel of markers that could be used for clinical diagnosis. Further exploration is needed to verify our previous results.As the results showed, the methylation level of  samples . Moreoved in RCC ,21,35,36CCND2 is down-regulated in renal cell cancer strongly suggests that the function of CCND2 is not only limited to its role in cell cycle transition from G1 to S. The cancer-specific loss of CCND2 in renal carcinoma and suppression of RCC cell growth provide clues for investigating a possible role of CCND2 in carcinogenesis in kidney and the methylation of its promoter may function as an optional biomarker for clinical application.In conclusion, the result that


Cellular responses to proton beam irradiation are not yet clearly understood, especially differences in the relative biological effectiveness (RBE) of high-energy proton beams depending on the position on the Spread-Out Bragg Peak (SOBP). Towards this end, we investigated the differences in the biological effect of a high-energy proton beam on the target cells placed at different positions on the SOBP, using two human esophageal cancer cell lines with differing radiosensitivities.10 for each position of the target cell lines on the SOBP was determined based on the results of the cell survival assay conducted after photon beam irradiation. In addition, the number of DNA double-strand breaks was estimated by quantitating the number of phospho-histone H2AX (γH2AX) foci formed in the nuclei by immunofluorescence analysis.Two human esophageal cancer cell lines  with different radiosensitivities were irradiated with a 235-MeV proton beam at 4 different positions on the SOBP , and the cell survivals were assessed by the clonogenic assay. The RBEIn regard to differences in the RBE of a proton beam according to the position on the SOBP, the RBE value tended to increase as the position on the SOBP moved distally. Comparison of the residual number of γH2AX foci at the end 24 h after the irradiation revealed, for both cell lines, a higher number of foci in the cells irradiated at the distal end of the SOPB than in those irradiated at the proximal end or center of the SOBP.The results of this study demonstrate that the RBE of a high-energy proton beam and the cellular responses, including the DNA damage repair processes, to high-energy proton beam irradiation, differ according to the position on the SOBP, irrespective of the radiosensitivity levels of the cell lines.The online version of this article (doi:10.1186/s13014-017-0849-1) contains supplementary material, which is available to authorized users. Proton Beam Therapy (PBT) has been used clinically, with or without concurrent chemotherapy, for several cancers, including locally advanced non-small cell lung cancer or esophageal squamous cell carcinoma (ESCC) –5. PBT iIn the application of PBT to clinical cancer treatment, we routinely adopt a Relative Biological Effectiveness (RBE) of 1.1. The RBE is approximated based on reported results from in vivo and in vitro experiments using a hamster non-tumor cell line and rodent cancer cell lines –9. EvaluWhile the RBE has been regarded as being constant throughout the range of the SOBP, recent studies have suggested that the RBE of a 62-MeV proton beam increased as the position on the SOBP became more distal, along with increase of the linear energy transfer (LET) , 11. HowThe number of DNA double-strand breaks (DSBs), which are well-known to play a major role in cell killing, after proton beam irradiation, has been shown as a useful predictor of the tumor radiosensitivity , 19. HowTherefore, we investigated the differences in the RBE and the cellular responses, especially focusing on DNA-DSB repair processes, depending on the position of the target cells on the SOBP, in response to high-energy proton beam irradiation of the target cells using 2 ESCC cell lines with differing radiosensitivity levels.2 at 37 °C.The human esophageal cancer cell lines OE21 and KYSE450 were obtained from the cell banks of Public Health England  and the National Institutes of Biomedical Innovation , respectively, and used within 20 passages for the present experiments. Both the cell lines were authenticated for their identity by Short Tandem Repeat analysis. The cell lines were selected based on the results of preliminary experiments carried out using 6 cell lines that showed different sensitivities to a photon beam, i.e., the OE21 cells were moderately sensitive to a photon beam, while the KYSE450 cells showed a low sensitivity . For the photon irradiation, both cell lines were irradiated with a 6-MV x-ray beam at the dose rate of 6 Gy/min, using Linac . For the proton irradiation, both cell lines were irradiated with a 235-MeV proton beam . Proton beams dispersed by a double-scattering method were shaped down with a brass collimator to irradiate a field size of 20 cm × 20 cm. The depth of irradiation was precisely modulated by placing polyethylene plates of appropriate thickness, considering the water equivalent thickness of 3 mm calculated by the thickness of the flask or dish and medium on the incident side, at a position between the collimator and the samples. The field size was 15 cm × 20 cm and the flask surface dose homogeneity was ≥95%. To evaluate the difference in the cellular responses to the proton beam irradiation according to the position on the SOBP, the cells were irradiated at 4 different positions on the SOBP, which was 8 cm in width (position #1) At entry (49 mm); position # 2) Just proximal to the SOBP (107 mm); position #3) Center of the SOBP (135 mm); position #4) Distal end, at the fall-off, of the SOBP (163 mm), at the dose rate of 6 Gy/min. The dosimetry error in the absolute dose was estimated to be a maximum of 1% at the center of the SOBP. The dose distribution within the irradiation field was also examined and the error range was 1% to 5% at the center of the SOBP.2 flasks  or 35-mm μ-dishes , and placed on polyethylene plates at the time of irradiation of the perpendicular proton or photon beam. All experiments were conducted in triplicate.The cells were seeded onto 25-cm2 tissue culture flasks containing 5 ml of the culture medium at 400 to 1600 cells per flask, depending on the irradiation dose, and incubated for 24 h prior to the irradiation. The flasks were irradiated with the proton or photon beams and returned to the CO2 incubator at 37 °C. After 8 or 12 days, the colonies were fixed with 4% formalin solution and stained with 1% crystal violet. Colonies that contained more than 50 cells were counted, and the surviving fractions were calculated as the ratio of the plating efficiencies of the irradiated to unirradiated cells. Cell survival curves were fitted to a linear-quadratic model:The OE21 and KYSE450 cells were seeded in triplicate onto 25-cmhttps://root.cern.ch/). The RBE10 and RBE37 (doses required to obtain a reduction in the fraction of the surviving cells to 10% (D10) and 37% (D37), respectively), relative to that of a 6-MV photon beam, were calculated for each position on the SOBP [where S represents the surviving fraction, and D represents the dose of radiation, α and βare adjustable parameters. Non-linear regression analysis was carried out using the ROOT software  and fixing with 4% formaldehyde for 15 min at each time-point. They were then washed again with PBS three times for 5 min each and blocked with blocking buffer containing 1% filtered bovine serum albumin  and 1% TritonX-100  in PBS, followed by incubation for 1 h at room temperature. Thereafter, the cells were incubated with anti-rabbit antibody directed against phospho-histone H2A.X Serine139  overnight at 4 °C. After incubation with the primary antibody, the cells were washed again three times with PBS, followed by the addition of blocking buffer containing goat anti-rabbit Alexa Fluor 488 IgG secondary antibody  was added; the cell suspensions were then left to stand for 2 h at room temperature. Thereafter, the cells were washed again with PBS 3 times before the addition of mounting medium containing DAPI . The experiments were performed in triplicate. Images were obtained under the Carl-Zeiss LSM710 confocal fluorescence microscope. We randomly examined six to nine microscopic fields for γH2AX (green) foci and nuclei stained with DAPI (blue).Cells were seeded onto 35-mm μ-dishes at the density of 1.0 × 10https://imagej.nih.gov/ij/) and the mean number of foci per nucleus was calculated. More than 100–200 cells were evaluated after the irradiation.The γH2AX foci in each cell were counted using the National Institutes of Health Image J software , and also between position #2 and position #4 (p = 0.012). The surviving fraction of the KYSE450 cells following irradiation at the dose of 6 Gy was significantly between position #1 and position #3 or position #4 (p = 0.002), and between position #2 and position #3 or position #4 (p = 0.031); furthermore, the surviving fraction of the cells following irradiation at 8 Gy was also significantly different between position #1 and position #3 or position #4 (p = 0.003), and between position #2 and position #3 or position #4 (p = 0.007) , and a trend towards a higher RBE than the routinely used fixed RBE value of 1.1 at the distal end of the SOBP. The differences in the RBE10 and RBE37 in the OE21 cells between position #2 and position #4 were 6.0% and 5.4%, respectively. The differences in the RBE10 among the positions on the SOBP were also significant  in the KYSE450 cells. Furthermore, the differences in the RBE10 and RBE37 in the KYSE450 cells between position #2 and position #4 were 24% and 17%, respectively. Consistent with these findings, the RBE10 and RBE37 of both cell lines were significantly different between beam entry and positions on the SOBP, and the differences among positions within the SOBP were also over 5%.The survival curve after photon irradiation showed that the shoulder of the survival curves was larger for the OE21 cells  than for the KYSE450 cells  , while the correlation between the RBE37 and LET in the KYSE450 cells (p = 0.04) was statistically significant.Figure Additional file p = 0.000, p = 0.002, and p = 0.007, respectively). A similar trend was noted in the KYSE450 cells. There were significant differences in the number of γH2AX foci in the cells at 24 h after irradiation between position #1 and position #4 and between position #2 and position #4 .Figure 2 = 0.76 and R2 = 0.99, respectively). Additional file The correlation between the surviving fraction after irradiation at 8 Gy (SF8) and the residual number of γH2AX foci/cell at 24 h after irradiation is shown in Fig. The RBE tended to be higher at the distal end of the SOBP than that at the proximal portion of the SOBP, irrespective of the radiosensitivity of the cell lines used. In addition, the amount of unrepaired double-stranded DNA breaks, as assessed by the number of γH2AX foci formed in the cells, at 24 h after irradiation was higher for irradiation at the distal end of the SOBP. Our findings indicate that the RBE as well as the cellular responses, including DNA damage repair processes, in response to proton beam irradiation differed according to the position on the SOBP.The RBE of proton beam irradiation against two ESCC cell lines of differing radiosensitivities in this study differed according to the position on the SOBP, and varied in the range of 1.03 to 1.40. Previous reports have shown differences in the RBE, ranging from 1.3 to 2.8, according to the depth, namely, position on the SOBP, for lower-energy proton beams (62–87-MeV) , 12, 13.Evaluation of the differences in the RBE depending on the position along the SOBP in previously reported studies has mainly been conducted using rodent cells and human malignant tumor cells  cells, human cervical carcinoma (HeLa) cells, head and neck squamous carcinoma (SQ20B) cells, and human melanoma HTB140) cells [0 cells , 23. HowDifferences in the RBE for irradiation targets in different positions along the SOBP would also be expected to influence the cellular responses to the proton beam. In explaining the mechanism underlying the correlation between the RBE and the cellular responses, LET has an important role. LET has been demonstrated to increase steeply at the distal end of the SOBP , 20, 24.When DSBs occur in cells exposed to photon- or proton-beam irradiation, formation of γH2AX foci represents one of the earliest events of the DNA damage repair process; these foci appear within minutes and reach their peak number at 0.5 h . In the In contrast, the residual number of γH2AX foci at 24 h after the irradiation was significantly higher for position #4  of the SOBP in both cell lines.The residual number of γH2AX foci has been interpreted as being reflective of persistent unrepaired DNA damage and as a predictor of the tumor radiosensitivity , 19, 29.The residual number of γH2AX foci is usually increased by high-LET irradiation. Bracalente et al. showed that the residual number of γH2AX foci at 6 h after high-LET irradiation was higher than that after γ-ray irradiation in a Chinese hamster ovary cell line . AntonelIn the clinical setting, we often select the physical proton dose based on the RBE of 1.1, assuming homogeneity along all positions of the SOBP. However, our current study demonstrated that the RBE differed according to the position on the SOBP. Moreover, the differences in the RBE among the positions on the SOBP in both cell lines were 5% or more . Thus, it may be necessary to take into consideration an inhomogeneous distribution of the RBE in the range of the SOBP for treatment planning in patients scheduled to undergo PBT. Further clinical studies are needed to clarify how the inhomogeneity of the RBE within the range of the SOBP would affect the clinical outcomes.Our study had the following limitations. First, we evaluated only 2 cancer cell lines. The RBE and cellular responses to proton beam irradiation could differ depending on the cell line used. Therefore, caution should be exercised in extrapolating the results of this study to other type of tumors or normal cells. Second, it seemed that the number of γH2AX foci at 0.5 h after irradiation of 8Gy using the National Institutes of Health Image J software was low. Thus, further analysis using another technique, such as flow cytometry, would be needed. Third, the current study was performed in vitro. In order to apply the results to clinical treatment, investigations in vivo would be needed, because the microenvironments of tumors and/or normal tissues could be expected to affect the RBE and cellular responses to proton beam irradiation. Thus, further investigations are warranted to confirm the validity of the results of this study. In addition, while PBT has been used with concurrent chemotherapy for locally advanced cancers, such as locally advanced non-small cell lung cancer, in clinical practice, there are few radiobiological reports regarding the effect of PBT used in combination with chemotherapy. Therefore, we should clarify the influence of differences in the radiosensitizing effects of the chemotherapeutic agents used on the differences in the effects of proton beam irradiation depending on the position on the SOBP.In this study, we demonstrated that the RBE of a high-energy proton beam and the cellular responses, including the DNA damage repair processes, to irradiation with a high-energy proton beam, differed according to the position on the SOBP, regardless of the radiosensitivity levels of the cells, in two human cancer cell lines.Additional file 1: Figure S1.Survival curves of 6 different esophageal squamous cell carcinoma cell lines irradiated with a 150-kV photon beam in preliminary experiments. Each experiment was conducted in triplicate. Error bars indicate the standard error of the mean. (TIFF 343 kb)Additional file 2: Figure S2.p < 0.05, **p < 0.01, ***p < 0.001) (TIFF 337 kb)Surviving fraction following irradiation at 6 Gy and 8 Gy in the OE21 and KYSE450 cells. Error bars indicate the standard error of the mean.  antibody and DAPI (blue). (TIFF 2421 kb)Additional file 4: Figure S4.2 = 0.81 and R2 = 0.79, respectively). (TIFF 225 kb)Correlation with the mean number of foci per cell at 24 h after the irradiation and the linear energy transfer (LET). The clear and filled circles show the results for position #1 and position #2, respectively. The clear and filled squares show the results for position #3 and position #4, respectively. Error bars indicate the standard error of the mean. A strong positive correlation between the mean number of foci formed per cell and the LET was observed in both the OE21 and KYSE450 ells (R


During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the  Despite the enthusiasm generated by these drugs, there is still much to be improved regarding their homogeneity, efficacy and safety  wer wer20] wpression . Similarnjugates , only a 2 to a variety of antibodies that incorporated engineered cysteine residues  were evaluated (ADC4-11) were uncapped by first reducing the engineered cysteines and the interchain disulfides using excess TCEP followed by the re-oxidation of the hinge cysteines using dehydroascorbic acid (DHA) and treatment with excess linker-payload 2. After workup and purification, the DAR of the resulting site-specific ADCs ranged from 1.7–2.0. Similar to the hinge cysteine conjugates, the site-specific single cysteine based ADCs showed excellent potency against high Her2 expressing N87 cell lines . The relative exposure of the conjugated payload to bulk solvent was assessed by hydrophobic interaction chromatography, as previously described [The cytotoxicity of ADCs that were generated by site-specific conjugation of the thialanstatin LP valuated . Auristavaluated , ADC4-11in situ and subsequently conjugated to a model antibody (4) Concurrently, we generated a two-payload carrying azido peptide by conjugating the lysines of the N-terminal azidoacetyl substituted peptide (SKGSK) with an N-hydroxysuccinimide (NHS) ester of PEG5-MMAD (5). A small, fairly neutral 5-mer peptide with two lysines separated by glycine-serine was chosen to reduce any potential instability. The excess NHS ester was quenched with ethanolamine and the resulting double-payload carrying peptide linker (7) was then conjugated to the DBCO-preprogrammed antibody (6) via an in situ copper free click reaction thus yielding a site-specific ADC with a DAR of 3.8 (ADC11). This ADC was purified using size-exclusion chromatography (SEC) and characterized by LC-MS and analytical SEC. The cytotoxicity of ADC11 was measured against cancer cell lines with various levels of Her2 expression and the ADC was found to be extremely potent against Her2 expressing N87 (IC50 = 0.49 nM) and 361 (IC50 = 0.39 nM) cell lines and exhibited approximately 1000 fold selectivity against the non-Her2 expressing 468 cell line. By comparison, a DAR4 lysine conjugate of 5 (not shown) exhibited an IC50 of 0.95 nM against N87 cells and 0.16 nM against 361 cells.As we have previously shown that the potency of lysine thailanstain ADCs  are highantibody  by clickantibody , 4 ConcuEG5-MMAD , 5. A smR of 3.8 , ADC11. 8) to yield a double thailanstatin conjugated azidopeptide. This dual-loaded peptide was then conjugated to four different DBCO preprogrammed single cysteine mutant trastuzumab antibodies using copper free click conjugation  exhibited low nM activity against the 361 cell line followed by ADC13 (392C) > ADC12 (114C) > ADC15 (k183C).Having demonstrated the ability to load an auristatin payload using the MPP chemistry, the N-terminal azidoacetyl SKGSK peptide was conjugated to the NHS ester of thailanstatin A , 8 to yijugation . This mejugation  one highell line . SurprisADC14 and ADC13, respectively) in the nude mouse N87 xenograft tumor model. The mice were injected with ADCs intravenously at two different doses of 1.5 mg/kg and 3 mg/kg as described previously. While at the 1.5 mg/kg dose the ADCs were only marginally efficacious . Gratifyingly, this double mutant showed much improved activity against moderate Her2 expressing 361 cell lines and maintained potent cytotoxicity towards the N87 cell lines in the in vitro cytotoxicity assay. To assess if the activity against the moderate antigen expressing cell lines is not just a characteristic of higher loaded site-specific ADC, we evaluated the cytotoxicity of four more ADCs (ADC17-20) generated by other combinations of engineered cysteine sites, 347+k183, 388+421, 347+392 and 443+k183. Two of the ADCs that were generated on antibodies where at least one site of conjugation was either 443 or 392 (ADC19 and ADC20) exhibited improved cytotoxicity against the 361 cell lines, as compared to the other two ADCs that were not conjugated at these preferred sites (ADC17 and ADC18)  .ADC16) in an N87 tumor xenograft bearing nude mouse study as described above. Three doses  were administered (IV) and a dose dependent tumor regression by this site-specific ADC was observed  ADCs which continue to suffer from poor therapeutic efficacy in the clinic [Thailandensis burkholderia MSMB43 that exerts its activity by binding to the SF3b subunit of the U2 snRNA subcomplex of the spliceosome [T. burkholderia that allows for gram-scale production of thailanstatin A [50 values against multiple cancer cell lines. Overexpression of MYC, an aggressive oncogene, is one of the most common drivers of human cancer and MYC dependent cell lines have remained resistant to therapeutics. Recently, Westbrook et al. reported that in vivo inhibition of the spliceosome impairs tumorigencity of MYC-dependent breast cancers and their metastatic potential [Despite the success of ADCs like ADCETRISe clinic . One appe clinic , 27. In e clinic , 18. We iceosome , 28, 29.statin A . The mecotential . Thus, ootential , 32 due 50 values of the resulting hinge-cysteine ADCs were very high against the 361 cell lines in spite of their high DARs (6.3 to 7.0). Despite lower potency against the moderate Her2 expressing 361 cell lines, we wanted to evaluate these ADCs in vivo in an N87 gastric cancer xenograft model due to their potent in vitro activity against the N87 cell line. Therefore, one of the thailanstatin ADCs (ADC1) which displayed excellent in vitro potency against a high Her2 expressing N87 cell line was selected for in vivo evaluation. Dosed at 3 mg/kg (qdx4), this ADC exhibited only marginal activity in an N87 gastric cancer xenograft model compared to the vehicle control  and were selective against the non-antigen expressing cell lines (IC50’s of >1000 nM). However, none of the ADCs were very potent against the moderate Her2 expressing 361 cell line, exhibiting IC50’s of > 1000 nM , was conjugated to the two lysines of a peptide (SKGSK) derivatized with an azidoacetyl group at the N-terminus. The resulting double-MMAD loaded azido-peptide (7) was conjugated in situ to trastuzumab A114C that had been pre-loaded with a maleimide DBCO (6). This four loaded site-specific MPP ADC (ADC11) showed potent cytotoxicity against the antigen expressing cell lines and was selective against non-antigen expressing cell lines . In vitro cytotoxicity against the moderate antigen expressing 361 cell line indeed displayed excellent potency with much improved IC50 values while maintaining potency against the N87 cell line and selectivity against the non-expressing 468 cell lines (ADC1). This data clearly suggests that site of conjugation can play a critical role in the enhancement of thailanstatin ADC cytotoxicity.Based on the improved potency and efficacy of the 443 and 392 MPP ADCs, we hypothesized that a double-cysteine ADC generated by conjugating thailanstatin iodoacetamide payload directly to the 443 and 392 sites might show a similar level of improvement in efficacy. Therefore, a double cysteine mutant of trastuzmab 443 + 392 was generated and linker-payload ll lines . This isll lines , ADC1. TADC19 and ADC20, ADC16), a loading of four linker-payloads was obtained for these double-cysteine mutants. The conjugates derived from both trastuzumab (T)-347C+k183C (ADC17) and T- 388C+421C (ADC18) were not very potent against the 361 cell lines, whereas T-347C+392C (ADC19) and T-443C+k183C (ADC20), both of which carry one of the preferred sites, exhibited slightly improved potency against the 361 cell line . This ADC is also cytotoxic against cell lines that are resistant to the clinically approved ADC, T-DM1 [ADC16 was over 100 times more potent than T-DM1  linker ADC allowed us to screen for these preferred conjugation sites by increasing the DAR on a single site of attachment. MPP ADCs and the double-cysteine mutant ADC generated on these sites showed substantially improved tumor regression in comparison to the approved drug T-DM1  was dialyzed into Dulbecco’s Phosphate Buffered Saline . The dialyzed antibody is reduced with addition 5 equiv of tris(2-carboxyethyl)phosphine hydrochloride  and diluted to 15 mg/mL final antibody concentration using DPBS, 5 mM 2, 2', 2", 2‴-tetraacetic acid (EDTA), pH 7. 0. The reaction is incubated at 37°C for 1.5 h and then cooled to room temperature. Conjugation was performed in 50 mM borate buffer, pH 8.5 by addition of 11 equiv of iodoacetamide thailanstatin payload (1–3)  that was2 that was dissolved as a 10 mM solution in DMA. The conjugation was performed at 10 mg/mL for 16 h at room temperature. The ADC was purified and characterized as described earlier [Site-directed mutagenesis, antibody expression, and purification were performed as previously described . The ant earlier . Briefly50 values were calculated using a four-parameter logistic model with XLfit  and are the arithmetic mean of multiple determinations.Cell lines and Cytotoxicity Assay: N87 (gastric cancer) and MDA-MB-468 (breast cancer) cell lines were obtained from ATCC ; MDA-MB-361-DYT2 (breast cancer) cells were generously provided by Dr. Dajun Yang at Georgetown University. TDM1-resistant MDA-MB-361-DYT1-TM and N87-TM cells were generated as described previously , 24. N876 of N87 cells respectively in 50% Matrigel . Mice were randomized into study groups when tumors reached approximately 150 mm3 to 300 mm3. Either phosphate buffered saline , trastuzumab-thailanstatin ADCs with different DAR, or PT-DM1 at different doses were administered intravenously starting on day 0 for a total of four doses, 4 days apart (Q4d x4). Tumors were measured at least weekly with a calibrator  and the tumor mass was calculated as volume = (width X width X length)/2.All animal studies were approved by the Pfizer Institutional Animal Care and Use Committee according to established guidelines. Female athymic nu/nu  were injected subcutaneously in the flank with suspensions of 1 X 104; Sigma-Aldrich, catalog no. 760676) was performed as described earlier.[7) was prepared by reacting 2mM aizoacetyl-Ser-Lys-Gly-Ser-Lys  with 8 mM NHS-ester payload [7 was reacted with 5–10 mg/mL of 6 in DPBS for 3h at 37°C. The resulting MPP ADC was then purified and characterized as described above.Conjugation of cysteine-mutant antibody with Malemide-PEG4-DBCO Click here for additional data file.S1 FigADC14 in N87 gastric cancer xenograft model dosed at 3 mg/kg (q4d x 4) showing three complete regression.(A) In vivo efficacy of MPP ADC 13 and MPP ADC14 in N87 gastric cancer xenograft model dosed at 1.56 mg/kg (q4d x 4). (B) Individual mouse in vivo efficacy of MPP ADC13 in N87 gastric cancer xenograft model dosed at 3 mg/kg (q4d x 4) showing two complete regression. (C) Individual mouse in vivo efficacy of MPP (PDF)Click here for additional data file.S2 FigADC16 in N87 efficacy model.(A) Individual mouse in vivo efficacy of MPP-ADC 16 in N87 gastric cancer xenograft model dosed at 3 mg/kg (q4d x 4) showing three complete regression. (B) Body weight data in grams for mice treated with 3 mg/kg (q4d x 4) of (PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Klotho, is a transmembrane protein, performs as a circulating hormone and upstream modulator of the insulin-like growth factor-1 receptor (IGF-1R), fibroblast growth factor (FGF), and Wnt signaling pathways. These pathways are involved in the development and progression of B cell lymphoma. We aimed to explore the expression pattern and functional mechanism of Klotho in diffuse large B cell lymphoma (DLBCL).Immunohistochemistry (IHC) and western blotting were performed to detect the expression level of Klotho in DLBCL patients and cell lines. Tumor suppressive effect of Klotho was determined by both in vitro and in vivo studies. Signaling pathway activity was assessed by western blotting.Remarkable lower expression levels of Klotho were observed in DLBCL patients and cell lines. Enforced expression of Klotho could significantly induce cell apoptosis and inhibit tumor growth in DLBCL. Upregulation of Klotho resulted in declined activation of IGF-1R signaling, accompanied with decreased phosphorylation of its downstream targets, including AKT and ERK1/2. Moreover, xenograft model treated with either Klotho overexpression vector or recombinant human Klotho administration presented restrained tumor growth and lower Ki67 staining.Our findings establish that Klotho performs as a tumor suppressor and modulator of IGF-1R signaling in DLBCL. Targeting Klotho may provide novel strategies for future therapeutic intervention. Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL), accounts for nearly 40% of all newly diagnosed cases . This diKlotho is an anti-aging gene originally identified in 1997 . Kuro-o The tumor suppressive activity of Klotho was first identified in breast cancer in 2008 . Recent A large number of aberrant receptor tyrosine kinases (RTKs) have been found in hematological malignancies , 17, butIn this present study, we aimed to assess the expression level and functional mechanism of Klotho in DLBCL. We identified reduced expression of Klotho in DLBCL for the first time. Noted inhibition of cell growth and induction of apoptosis were observed in DLBCL with Klotho overexpression. Tumor growth was restrained by administration of Klotho protein in xenograft model of DLBCL. Our findings demonstrated that Klotho was a tumor suppressor and modulator of IGF-1R signaling in DLBCL, indicating that targeting Klotho may provide novel therapeutic strategy in DLBCL.This study was approved by the Medical Ethical Committee of Shandong Provincial Hospital affiliated to Shandong University. The paraffin-embedded archived samples from 50 newly diagnosed DLBCL patients and 20 reactive hyperplasia patients were collected. Samples of patients with reactive hyperplasia were referred as control. Histological diagnoses were established according to the WHO classification . PeripheHuman DLBCL cell lines LY1 and LY8 were cultured in Iscove modified Dulbecco medium with 10% heat-inactivated fetal bovine serum. The medium contains 1% penicillin/streptomycin mixture and 2 mmol/l glutamine. CD19+ B cells and PBMCs obtained from three healthy donors were used as controls . Recombinant human Klotho (rhKL) and recombinant human IGF-1 were obtained from PeproTech , and adriamycin (ADR) was bought from Actavis .Lentivirus vectors either encoding Klotho or an empty lentiviral vector were from Genechem . Lentivirus transfection was carried out according to the manufacturers’ instruction. Infection efficiencies were assessed by green fluorescent protein (GFP) through flow cytometry. The stably transfected cells were selected 48 h later with 5 μg/ml puromycin .△△CT method.Total RNA was extracted using RNAiso Plus . Reverse transcription reaction was conducted with the reverse transcription reagents . Amplification reactions were performed with SyberGreen  in LightCycler 480II . Klotho-specific primers were as follows: forward, 5′-AGCAATCTGGTCTGAATAACACTGG; reverse, 5′-CATGTTTCAGCGTGAAAGTTCAAAG. Relative quantification was calculated using the The 4-μm-thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized and hydrate. Antigen retrieval was performed using 0.01 mol/l sodium citrate buffer (pH 6.0) under high pressure followed by a 1-h cool-down and rinses in phosphate buffer solution (PBS). Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 15 min, followed by incubation with normal serum to block non-specific binding. The slides were then incubated overnight at 4 °C with primary antibodies, anti-Klotho (1:150) or anti-Ki67 (1:100). After washing, the tissue sections were treated with the second antibody from SP reagent kit  for 30 min at room temperature, followed by further treated with strept avidin-horseradish peroxidase complex (SABC) for 30 min at room temperature. After treated with diaminobenzidine (DAB) Kit , the stained slides were counterstained with hematoxylin and mounted. Negative control was carried out with the primary antibody replaced by PBS. IHC staining was scored by the proportion of positive tumor cells. Five microscopic fields with the highest immunoreactivity at ×400 magnification were evaluated by two independent observers who were blinded to the patients’ clinical data. Cases with at least 10% of tumor cells with Klotho staining were considered as positive. Fresh mice subcutaneous tumors were fixed in 4% paraformaldehyde (PFA) and embedded with paraffin for histological examinations. Sections with 4-μm thickness were cut and stained with hematoxylin-eosin (H&E).Cells were lysed in radio-immunoprecipitation assay buffer  together with 1× phosphatase inhibitor cocktail . The BCA assay  was performed to detect protein concentration. Protein extracts (30 μg) were then electrophoresed on SDS-polyacrylamide gels and blotted from the gel onto polyvinylidene fluoride membranes . Membranes were incubated with the blocking solution  for 1 h at room temperature and then immunoblotted with the indicated antibodies (1:1000 dilution) overnight at 4 °C. After which, the membranes were washed with TBS-T and then probed with the HRP-conjugated secondary antibodies  and the electro-chemi-luminescence kit . Chemiluminescent signals were detected using the Amersham Imager 600 imaging system . ImageJ software  was used to quantify the protein bands normalized to control. Primary antibodies used were Klotho , phospho-IGF-1R (Tyr1135/1136), IGF-1R, phospho-AKT(Ser473), total pan-AKT, Mcl-1, Caspase-3, diphosphorylated and total ERK1/2 , β-actin, and GAPDH . The experiments were performed in triplicate with GAPDH or β-actin  as endogenous control.Cell proliferation was assessed by performing triplicate assays with the Cell Counting Kit-8 (CCK-8) assay . DLBCL cells with designed treatment were seeded in 96-well plates at a density of 5000 cells/well for 48 or 24~96 h later. Thereafter, the cells were incubated with 10 μl/well CCK-8 for 4 h according to the manufacturer’s proposal. Cell proliferation was detected by light absorption at 450 nm by Multiskan GO Microplate Reader .−/7AAD−), dead cells (Annexin V-PE−/7AAD+), apoptotic cells (Annexin V-PE+/7AAD−), and necrotic cells (Annexin V-PE+/7AAD+). The rates of apoptotic cells were acquired on a FACS-Navios Flow Cytometer . Data were analyzed using FlowJo Version 7.6 software .Apoptosis of transfected DLBCL cells were detected by Annexin V-PE/7-aminoactinomycin (7AAD)  assay according to the manufacturers’ instructions. DLBCL cells with designed treatments were harvested and washed twice with ice-cold PBS and incubated in 1× binding buffer (containing 5 μl Annexin V-PE and 5 μl 7AAD). After incubation in the dark for 15 min, cells were subjected to the flow cytometry. At least 10,000 events per sample were acquired. Cells were discriminated into viable cells , with a lower limit of assay of 6.15 pg/ml.7 LY1 cells , mixed with 100 μl Matrigel , were subcutaneously injected into the right inferior limb of mice. Tumor size was measured by the digital caliper. For the experiment with rhKL, SCID Beige mice were injected subcutaneously with 1 × 107 LY1 cells into the left inferior limbs. Mice were treated with daily intraperitoneal injections of rhKL (7.5 μg/kg) or PBS control (six mice per group) for 2 weeks. The volume of tumor was estimated using the equation V = (a × b2) × 0.5236, where a is the largest dimension and b is the perpendicular diameter.All animal experiments were performed in accordance with the principles of the Institutional Animal Care. Severe combined immunodeficiency (SCID) Beige female mice of 5-week old were bought  and maintained in a pathogen-free environment under controlled condition of light and humidity. 1 × 10t tests were used to assess the differences between groups. p < 0.05 was considered to be statistically significant.All statistical analyses were performed by using the statistic software SPSS17.0  for Windows. In vitro experimental results were presented as mean ± SD of data obtained from three separate experiments. Overall survival time was measured from the date of diagnosis to the date of death or the last follow-up. Kaplan-Meier analysis was performed to estimate the survival functions. A log-rank test was used to assess survival differences. Chi-square test was used to analyze the clinical characteristics of patients. One-way analysis of variance (ANOVA) or p = 0.002, Table n = 50). The median survival of patients in Klotho-positive group was 48.46 months, significantly longer than those in Klotho-negative group  or Klotho-overexpression lentivirus vectors (LV-KL). The upregulation of Klotho levels were confirmed by quantitative PCR and western blot Fig. . Cell prp = 0.002) and LY8 cells  or a control vehicle (n = 6) for 2 weeks. Significant decreased tumor volumes were observed in the mice treated with rhKL compared with those treated with vehicle control  than the control subjects (818.87 ± 241.51 pg/ml, In this study, our observations identified for the first time that Klotho, an anti-aging gene, as a potential tumor suppressor in DLBCL tumorigenesis. Klotho was downregulated in human DLBCL and inhibited the growth of DLBCL both in vitro and in vivo. Upregulation of Klotho resulted in declined activation of IGF-1R signaling pathway in DLBCL.We identified remarkable reduced expression of Klotho in DLBCL tissues and cell lines, whereas higher expression in reactive hyperplasia and CD19+ B cells from normal donors. Decreased level of Klotho was associated with advanced stage and more aggressive disease process in DLBCL. Tumor suppressive activity of Klotho has been reported in several human solid malignancies, but never in hematological cancers –33. ReceDLBCL is a high-grade heterogeneous disorder defined by heterogeneity in clinical and biological characteristics , 38, 39.As Klotho exists in both membrane-bound form and secreted form, the secreted Klotho could be shed and released into the circulation . Recent Importantly, our study illuminated that Klotho effectively inhibited the growth of DLBCL cells. Overexpression of Klotho significantly inhibited cell proliferation and induced cell apoptosis in DLBCL. We also discovered that efficacy of ADR could be enhanced by combination with rhKL in DLBCL cells. This finding suggested the potential of Klotho in therapeutic intervention of DLBCL. Our in vivo investigations demonstrated that upregulation of Klotho, either by LV-KL transfection or rhKL administration, congruously led to inhibitory effect in the tumor growth of xenograft model of DLBCL. This will pave the way for rational design of Klotho-based molecular products in DLBCL. Nevertheless, pharmacokinetic investigations are still required to explore the optimal dose and adverse reaction of rhKL in the use of either single agent treatment or drug combination.Significantly, we also found that Klotho could inhibit the activation of IGF-1R signaling in DLBCL. Induction of IGF-1R signaling was involved in the pathogenesis of hematological malignancies . RecentlTaken together, our findings identified that Klotho performs as tumor suppressor and modulator of IGF-1R signaling in the DLBCL. Overexpression of Klotho may be a predictive marker for favorable outcome in DLBCL. Klotho reinforces the response of DLBCL cells to chemotherapeutic drug. Being an endogenous circulating hormone, the secreted Klotho could function as an active form and inhibit the tumor growth effectively both in vitro and in vivo. This study illuminates Klotho as a potential target for future therapeutic strategies.


Lysine-Specific Demethylase 1 (LSD1) over-expression correlates with poorly differentiated neuroblastoma and predicts poor outcome despite multimodal therapy. We have studied the efficacy of reversible and specific LSD1 inhibition with HCI-2509 in neuroblastoma cell lines and particularly the effect of HCI-2509 on the transcriptomic profile in MYCN amplified NGP cells. Cell survival assays show that HCI-2509 is cytotoxic to poorly differentiated neuroblastoma cell lines in low micromole or lower doses. Transcriptional profiling of NGP cells treated with HCI-2509 shows a significant effect on p53, cell cycle, MYCN and hypoxia pathway gene sets. HCI-2509 results in increased histone methyl marks and p53 levels along with cell cycle arrest in the G2/M phase and inhibition of colony formation of NGP cells. Our findings indicate that LSD1 inhibition with HCI-2509 has a multi-target effect in neuroblastoma cell lines, mediated in part via p53. MYCN-amplified neuroblastoma cells have a targeted benefit as HCI-2509 downregulates the MYCN upregulated gene set. Neuroblastoma is the most common extracranial solid tumor in childhood. More than 650 cases of neuroblastoma are diagnosed each year in North America . The cliMYCN and ARID1B. A switch in the histone code from the repressive H3K27me3 mark to the activated H3K4me3 mark has been noted on the promoters of MYCN and ARID3B in high risk neuroblastoma  ab16898.#: 4390827.LSD1 SiRNA: Ambion Cat#1 – Ambion AM4611 – 50 uM concentration.Silencer – Negative Control siRNA


The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order  in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.To identify suitable cell lines for a mimetic system of  The physical barrier seals the paracellular passage of hydrophilic molecules while efflux transporters like P-glycoprotein (P-gp) restrict the transcellular passage of lipophilic molecules by extruding them back into the blood . BBB limin vivo BBB characteristics of tight junction and expression of efflux transporters. However, the primary cell culture is expensive, time-consuming and technique-demanding and thus the immortalized cell lines are developed for BBB permeability studies. Brain capillary endothelial cell lines such as mouse bEnd3, porcine PBMEC/C1-2 and human hCMEC/D3, and non-cerebral cell lines like CaCo-2, MDCK-MDR1 and ECV304 could form the tight paracellular barrier and represent popular cell lines for BBB studies [in vivo BBB may enhance the barrier function of endothelial cells. In addition, flow-based hollow-fiber models, microfluidic models and human pluripotent stem cells-derived models have been established for BBB studies. However, they require sophisticated expertises and are not in wide usage at present [Artificial membrane and cell culture models are common methods for BBB permeability studies. Parallel artificial membrane permeability assay (PAMPA), based on porcine brain extract, mimics the lipoidal microenvironment of BBB for passive diffusion transport . Primary studies . A co-cu present .ECV304, firstly reported as a human umbilical vein endothelial cell line and later proved to exhibit phenotypic characteristics similar to human bladder cancer cell line, is used for BBB studies due to its capacity to generate tight paracellular barrier , 8. It iTo identify cell lines suitable for drug testing of BBB permeability, we evaluated ECV304 and bEnd3 cell lines in the respect of barrier tightness and P-gp function. The ECV304 and bEnd3 monoculture and co-culture models with C6 cells were tested related to tightness through transendothelial electrical resistance (TEER) and permeability for Lucifer yellow. The tight junction proteins occludin, claudin-5 and ZO-1 on both cell lines were detected by immunofluorescence method. P-gp function was assessed using Rhodamine 123 (R123) uptake assay. Additionally, a permeability testing of reference compounds was performed on ECV304 and bEnd3 cell layers.ECV304, C6 and bEnd3 cell lines were obtained from American Type Culture Collection (ATCC), Medium 199 (M199), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin (0.25%)-EDTA (0.02%) solution, penicillin-streptomycin solution and Hank’s balanced salt solution (HBSS) were purchased from Hyclone , Lucifer yellow, quinidine, digoxin and R123 were purchased from Sigma Aldrich , verapamil and propranolol were obtained from Macklin Inc. .2 in air at 37°C and the confluent cells were passaged by the trypsin (0.25%)-EDTA (0.02%) solution at a split ratio of 1:5~1:10.ECV304 and C6 cell lines were incubated with M199 containing 10% FBS and 1% penicillin-streptomycin while bEnd3 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cell culture was performed in a humidified atmosphere of 5% CO4 and 8 × 104/cm2, respectively. For the generation of co-culture models, C6 cells were seeded on the lower surface of the membrane at a density of 5 × 104/cm2 and incubated for 2 h to allow cell attachment before the seeding of ECV304 or bEnd3 cells. The culture medium  was replenished every day.For the generation of mono-culture models, ECV304 or bEnd3 cells were seeded on the upper surface of the membrane in Polyester Transwell inserts  at a density of 5 × 102.TEER was measured using an Epithelial-volt-ohm-meter (EVOM) with Endohm-12 chamber electrodes . TEER of blank inserts was subtracted from the measured TEER of each model to reflect that of cell layers themselves. Values were expressed as Ω·cmOn day 5~7 after seeding cells, the TEER across cell layers was measured before initiating the transport study by replacing the medium in the apical compartment with tested compounds dissolved in HBSS. The permeability of Lucifer yellow (50 μM) was conducted on ECV304 and bEnd3 monoculture or co-culture models with C6 cells while those of other molecules  were performed on the monoculture models at 37°C. At indicated time points of 15, 30, 45 and 60 min, the inserts were transferred into new wells containing fresh HBSS to maintain sink conditions. Lucifer yellow  and R123  were measured by a PerkinElmer EnSpire Multimode Plate Reader. Other compounds were analyzed by liquid chromatography tandem mass spectrometer (LC-MS/MS).all) × surface area of the membrane insert . The PEblank of blank inserts was included to calculate the PEcell for cell layers themselves using the following equation: 1/PEcellS = 1/PEallS—1/PEblankS.The cleared volume was determined by dividing the compound amount in basolateral compartment by the compound concentration in apical compartment. The slope of the cumulative clearance versus time was calculated by linear regression analysis and denoted as permeability coefficient  for 10~20 min at room temperature. Then the cells were washed in PBS and permeabilized by 0.1% Triton X-100  in 5% normal goat serum  for 30min. The incubation with a 1:100 dilution of rabbit anti-occludin, ZO-1  and claudin-5  primary antibody was performed at 4°C overnight. After washing in PBS, the cells were treated with secondary antibody  for 30 min at room temperature and then stained with DAPI . The coverslips were mounted using fluorescent mounting medium  and the images were obtained by a ZEISS LSM 710 confocal microscope . Negative controls were performed in parallel in the absence of primary antibodies.The ECV304 and bEnd3 were seeded on the coverslips  at a density of 3×10R123 uptake assay was carried out to identify P-gp function. The confluent ECV304 and bEnd3 cells in 24-well plates were exposed to blank HBSS or HBSS containing P-gp inhibitor verapamil (100 μM) for 30 min at 37°C. Then the medium was removed and P-gp substrate R123 (5 μM) dissolved in HBSS alone or with the inhibitor was added to cells and the incubation lasted for 1 h. After replacing in HBSS with or without the inhibitor, the plate was maintained at 37°C for another 2 h. The cells were lysed with 1% Triton X-100 in PBS for 15min at 37°C and the aliquots were removed for R123 detection using a PerkinElmer EnSpire Multimode Plate Reader . The protein content was determined by a BCA assay kit . The R123 concentration was expressed as ng/mg protein.18 column  under gradient elution with mobile phase A (methanol) and mobile phase B (0.05% formic acid in water) as follows: initial, 10% A; 0 ~ 0.7 min 10% A– 90% A; 0.7 ~ 1.4 min, 90% A; 1.4 ~ 1.5 min, 90% A– 10% A; 1.5 ~ 3min, 10% A. The flow rate was 0.4 ml/min.The analysis was carried out on an Acquity UPLC H-class  tandem QTRAP 5500 mass system . The separation was operated on a Waters Acquity UPLC BEH Cm/z 325.1 > 172.1 for quinidine, m/z 455.2 > 165 for verapamil and m/z 260 > 183 for propranolol, respectively. The negative ESI was performed under ion spray voltage of -4500 V for digoxin (m/z 779.4 > 649.2).The positive electrospray ionization (ESI) was performed with optimal conditions as follows: ion spray voltage, 5500 V; temperature, 550°C; curtain gas, 30 psi; ion source gas 1 and ion source gas 2, 50 psi. The mass spectrometric analysis was operated in multiple reaction monitoring (MRM) mode with ion transitions at t test. Changes were considered as statistical significance at p < 0.05.The values were compared using the analysis of two-tail paired Student’s 2 and 11.17 ± 0.87 × 10−6 cm/s, respectively. The bEnd3 monolayer was looser than ECV304, as reflected by the lower TEER (30 ± 2.83 Ω·cm2) and higher permeability to Lucifer yellow (23.68 ± 1.71 × 10−6 cm/s). However, when co-cultured with C6 cells, ECV304 showed a 25% reduction of TEER and 2.2-fold increase of permeability to Lucifer yellow, while a 34% reduction of TEER and 2-fold increase of permeability to Lucifer yellow were observed in bEnd3. These data indicated the deleterious effects of C6 cells on the integrity of ECV304 and bEnd3 cell layers.Transport studies were performed at 5~7 days after the seeding of cells because TEER and permeability to Lucifer yellow reached plateau phase at this period and increasing incubation time will not enhance the tightness of cell layers. As show in The tight junction proteins occludin, claudin-5 and ZO-1 are crucial for the restrictive paracellular barrier of BBB. We detected the presence of three tight junction proteins in ECV304 and bEnd3 cells by immunofluorescence method. The staining of ZO-1 was distinct on cell membrane while those of occludin and claudin-5 were diffused and weak .Efflux transporter P-gp is an important influential factor on drug permeation across BBB and R123 uptake assay was applied to assess its function in ECV304 and bEnd3 cells. R123 uptake in ECV304 was not significantly changed while that in bEnd3 cells was increased by 6.2-fold by the P-gp inhibitor verapamil versus absence of the inhibitor , which pThe permeability of reference compounds were validated in ECV304 and bEnd3 monoculture models. In both cell lines, the permeability of the tested compounds achieved a similar rank order: the lowest permeability was observed for digoxin and the highest permeability was obtained for propranolol. A higher permeability of R123 was determined versus that of digoxin. Quinidine and verapamil demonstrated the permeability close to that of propranolol. However, the permeability coefficients of the tested compounds across ECV304 were lower with different degrees than those in bEnd3 probably due to the tighter cell layers of ECV304 versus bEnd3 . In addiin vitro, we chose two immortalized cell lines, ECV304 and bEnd3 as their permeability are comparable to those of the in vivo BBB [To investigate a mimetic BBB model for drug permeability study vivo BBB , 12, and2 vs 62 Ω·cm2 and 11.17 × 10−6 cm/s vs 10.4 × 10−6 cm/s) [The functional tightness of BBB models are often assessed by two methods: TEER measurement and tracer flux assay . TEER re−6 cm/s) . In compIt is known that the astrocytes or glioma cells induce and strengthen BBB functions of brain endothelial cells via a range of released factors, including transforming growth factor-β (TGFβ), glial-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) –18. HoweTight junction is formed by a complex of tight junction proteins including transmembrane proteins occludin, claudin-5 and cytoplasmic protein ZO-1. Occludin and claudin-5 interact with membrane proteins on adjacent cells and form the backbone of tight junctions while ZO-1 links transmembrane proteins to actin cytoskeleton for the maintenance and regulation of paracellular barrier. Loss of these important tight junction proteins may result in increased barrier permeability or breakdown of BBB . Our stuP-gp is an extensively characterized efflux protein that influences the rate and extent of drug penetration into the brain by pumping compounds back into the blood. Molecular detection at mRNA or protein levels and functionality assays through cellular uptake experiment or bi-directional transport studies are explored to identify transport proteins . In thisDiscriminative brain penetration is an important feature of BBB and compounds with different permeability were evaluated in ECV304 and bEnd3 cells. In both cell lines, the lowest permeability value was observed for P-gp substrate digoxin and the highest permeability was obtained for lipophilic marker propranolol, which is consistent with previous studies in a triple-culture model of primary rat brain endothelial cells, astrocytes and pericytes . Since Ein vitro BBB permeability models with the TEER over 1000 Ω·cm2 and evident immunostaining of tight junction proteins [in vivo BBB tight barrier need to be carried out.The ECV304 developed tighter monolayers and better discriminated between transcellular and paracellular transport than bEnd3 while P-gp function was only identified in bEnd3. However, the monolayers of both cell lines lack the sufficient tightness of proteins . Further