A rapid and sensitive UPLC-MS/MS method for the determination of zanubrutinib in beagle plasma and its application in pharmacokinetics.

A reliable and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of zanubrutinib in the plasma of beagle dogs. The column used was an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm), maintained at 40°C with an injection volume of 2 µl. The gradient elution program was as follows: 0-1 min, 10-10% A; 1-1.1 min, 10-90% A; 1.1-2.1 min, 90-90% A; 2.1-2.2 min, 90-10% A; 2.2-3.0 min, 10-10% A. Mobile phase A was 0.1% formic acid, B was acetonitrile, and the total analysis time was 3 min. The mass spectrometry was performed in positive ion mode, and the scanning mode was multi-reaction monitoring mode with electrospray ionization as the ion source; m/z 472.2 → 455.01 for zanubrutinib and m/z 441.03 → 137.99 for ibrutinib (internal standard). The plasma samples were processed by protein precipitation. The standard curve showed good linearity (r2 = 0.999 8) in the range of 1.0-1,000 ng/ml (zanubrutinib) with a low limit of quantification of 1 ng/ml. Also, the intra-day and inter-day precision (RSD) was <5.88% and the accuracy (RE) ranged from -1.56 to 1.08%; the recoveries of zanubrutinib in beagle plasma ranged from 90.12 to 93.53% (RSD 1.67-6.42%) and the ME values of zanubrutinib were 98.70-101.06% (RSD 5.37-8.49%, n = 6). All values meet US Food and Drug Administration requirements. A rapid, highly selective and sensitive method for the determination of zanubrutinib concentration in plasma by UPLC-MS/MS was successfully developed. This method is suitable for pharmacokinetic studies in beagle dogs by following oral administration of zanubrutinib.

Simultaneous Determination of Five Components of Chaihu-Shugan-San in Beagle Plasma by HPLC-MS/MS and Its Application to a Pharmacokinetic Study after a Single Dose of Chaihu-Shugan-San.

Chaihu-shugan-san (CHSGS) has been widely used in China to treat depression and gastrointestinal diseases for thousands of years, but little is known about its pharmacokinetic properties. The purpose of our study is to develop a reliable and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to detect five components in beagle plasma and study their pharmacokinetic after oral administration of CHSGS in beagles. An Agilent C18 column (2.1 × 150 mm, 3.5 µm) was used to separate the analytes, and the column temperature was maintained at 40°C. A gradient elution procedure was used with solvent A (acetonitrile) and solvent B (0.1% formic acid, aqueous) as mobile phases. The elution procedure was 60% B-10% B (0-3 min) and 10% B-60% B (3.1-4 min). The flow rate was 0.3 mL/min, and the total measurement time was 4 min. Within the determined range, the standard calibration curves of the five analytes had a satisfactory linear relationship (r 2 ≥ 0.9923). The recovery rate (n = 6) of the five analytes was between 85.42% and 90.85%, and the matrix effects (n = 6) were between 94.52% and 103.91%. These results show that the validated method could be successfully applied to study the pharmacokinetic in beagles after a single dose of CHSGS.

Metabolomics analysis of the antidepressant prescription Danzhi Xiaoyao Powder in a rat model of Chronic Unpredictable Mild Stress (CUMS).

ETHNOPHARMACOLOGICAL RELEVANCE: Danzhi Xiaoyao Powder (DZXY) is a classical prescription, that has been extensively used in traditional Chinese medicine (TMC) to treat depression for many years. However, the mechanism of DZXY is still unclear. AIM OF THE STUDY: The aim was to investigate the mechanism of the antidepressant effect of DZXY on a rat model of chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: Forty male SD (Sprague-Dawley) rats with similar open field test (OFT) results were randomLy divided into a control group (n = 10) and an experimental group (n = 30). A depression model was established in the experimental group using the CUMS method. After the CUMS model was established successfully, the rats were randomLy divided into a depression model group and a DZXY group. The DZXY group was fed DZXY, while the depression model group and control group were given an equal amount of 0.5% sodium carboxymethyl cellulose suspension. Intragastric administration was performed once daily for 14 consecutive days. Animal weight, the sugar preference test, the open field test and the forced swimming test were used to evaluate the modeling effect and the antidepressant effect of DZXY. After the experiment, the plasma of rats was collected and the changes in plasma metabolites were analyzed by UPLC/Q-TOF-MS. The UPLC/Q-TOF-MS spectra data were evaluated by pattern recognition analysis to determine the changes in endogenous metabolites in the rat plasma samples. RESULTS: The results of the behavioral investigation showed that the rat model of depression was successfully replicated and that DZXY had an antidepressant effect. Using the UPLC-MS/MS metabolomics platform, partial least squares (PLS) and orthogonal partial least squares (OPLS), metabolic profile models (R2 and Q2 ≥ 0.5) of rat plasma were successfully constructed. The model could distinguish among the control group, the depression model group and the DZXY group. Finally, 38 differential metabolites were identified in the plasma. According to KEGG (http://www.kegg.jp) pathway analysis, amino acid metabolism, lipid metabolism, purine metabolism, the prolactin signaling pathway and bile secretion were enriched and represented the main metabolic pathways influenced in the plasma. CONCLUSIONS: This study successfully established a CUMS depression model. A total of 38 differential metabolites associated with depression were identified in the plasma of rats, 24 of which were modulated by DZXY. These results suggest that DZXY can improve excitability and play an antidepressant role by regulating phenylalanine metabolism, arachidonic acid metabolism, porphyrin metabolism, D-arginine and D-ornithine metabolism, steroid hormone biosynthesis, unsaturated fatty acid biosynthesis and steroid biosynthesis.

Development and Validation of UPLC-MS/MS Method for Determination of Enasidenib in Rat Plasma and Its Pharmacokinetic Application.

In our research, a straightforward UPLC-MS/MS method, with diazepam as the internal standard (IS), was proposed and acknowledged to determine the concentrations of enasidenib in rat plasma. When preparing the sample, we used acetonitrile for protein precipitation. The gradient elution method was used, and the mobile phase was acetonitrile and 0.1% formic acid. Diazepam was used as the IS. We used the Acquity UPLC BEH C18 column to separate enasidenib and IS. Under the positive ion electrospray ionization (ESI) source conditions, the mass transfer pairs of enasidenib were monitored by multiple reaction monitoring (MRM) to be m/z 474.2 ⟶ 456.1 and m/z 474.2 ⟶ 267.0, and the IS mass transfer pairs were m/z 285.0 ⟶ 154.0. Enasidenib had good linearity (r 2 = 0.9985) in the concentration range of 1.0-1000 ng/mL. Besides, the values of intraday and interday precision were 2.25-8.40% and 3.94-5.46%, respectively, and the range of the accuracy values varied from -1.44 to 2.34%. Matrix effect, extraction recovery, and stability were compliant with FDA approval guidelines in terms of bioanalytical method validation. We had established a new method that had been applied to the pharmacokinetic study of enasidenib in rats.

Simultaneous determination of six components of Danzhi Xiaoyao Pill in beagle plasma by HPLC-MS/MS and a study of pharmacokinetic of paeoniflorin and geniposide after single-dose administration.

This study was to develop a reliable and simple high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to detect paeoniflorin, geniposide, saikosaponin b2, liquiritin, paeonol and atractylenolide Ⅲ in beagle plasma and to study pharmacokinetic of paeoniflorin and geniposide after single-dose administration of Danzhi Xiaoyao Pill (DZXY). Chromatographic separation was performed using an Agilent C18 column, and multiple reaction monitoring (MRM) mode was used. A gradient elution procedure was used with solvent A (acetonitrile) and solvent B (0.1 % formic acid-water) as mobile phases. The elution procedure was as follows: 85 % B-30 % B (0-7 min) and 30 % B-30 % B (7.1-8 min). The flow rate was 0.3 mL/min. The column temperature was 40 ℃, and the injection volume was 10 µL. The main analytical parameters of paeoniflorin, geniposide, saikosaponin b2, liquiritin, paeonol and atractylenolide Ⅲ were m/z 525→449, m/z 433→224, m/z 780→617, m/z 417→254, m/z 167→43 and m/z 249→231, respectively. Ethyl acetate was used to extract the analytes in the plasma. Standard calibration curves of six analytes showed satisfactory linearity (r2≥0.99 2) within the determined ranges. The lower limits of quantification were 0.5 ng/mL for paeoniflorin and liquiritin, 2.5 ng/mL for geniposide and saikosaponin b2 and 1.0 ng/mL for atractylenolide Ⅲ and paeonol, respectively. The intra-day and inter-day precision (RSD %) were all below 6.94 %, and the intra-day and inter-day accuracy (RE %) were within ± 6.10 %. The recovery and ME of six analytes were 85.99 %-98.10 % and 95.78%-108.06%, respectively. Additionally, the method we established in this experiment can be successfully used to study the pharmacokinetics of paeoniflorin and geniposide in beagle plasma.

Simultaneous Determination of Parecoxib and Its Metabolite Valdecoxib Concentrations in Beagle Plasma by UPLC-MS/MS and Application for Pharmacokinetics Study.

A method for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagle plasma by UPLC-MS/MS was developed and validated. After the plasma was extracted by acetonitrile precipitation, the analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) using acetonitrile-formic acid as the mobile phase in gradient mode. The analytes were monitored by multiple reaction monitoring (MRM) in electrospray negative ion mode. The mass transfer pairs were m/z 368.97→119.01 for parecoxib, m/z 312.89→118.02 for valdecoxib, and m/z 379.98→316.02 for celecoxib (internal standard, IS). The correlation coefficients of parecoxib and valdecoxib ranged from 5 to 4000 ng/mL were greater than 0.9998. The recovery of parecoxib and valdecoxib was greater than 82.54%. The inter- and intra-day precision RSD values were 1.36~3.65% and 2.28~5.91%, respectively. The accuracy of RE values were -1.38%~1.96%. Finally, the matrix effect (ME) and stability were also within acceptable criteria. This method had been successfully applied to the pharmacokinetics of parecoxib and valdecoxib in beagle plasma after injection of parecoxib (1.33 mg/kg, intramuscular injection).

UPLC-MS/MS method for the determination of talazoparib in rat plasma and its pharmacokinetic study.

In the present study, an accurate and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of plasma talazoparib concentration in rats was developed and established. The purpose of chromatographic separation of talazoparib and the internal standard (bosutinib) was achieved on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with a flow rate of 0.40 mL/min, using a gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. The detection was performed on a XEVO TQ-S triple quadrupole tandem mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions of m/z 381.3 → 285.2 for talazoparib and m/z 530.2 → 141.2 for bosutinib (IS), respectively. The method was linear over the range of 0.5-200 ng/mL for talazoparib. The accuracies and precisions of intra- and inter-day were all within the acceptance limits, and no matrix effect was observed in this method. The validated method was further employed to a pharmacokinetic study of talazoparib after oral treatment with 0.2 mg/kg talazoparib to rats.

Effect Of Chinese Herb Danzhi Xiaoyao Pills On Pharmacokinetics Of Venlafaxine In Beagles.

OBJECTIVE: To investigate the effects of Chinese herb Danzhi Xiaoyao pills on the pharmacokinetics of venlafaxine and its metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in beagles by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: Six beagles (half male, half female) were chosen to test, being fasted before the experiment but having free access to drinking water 1 day before being fed drugs. After oral administration of venlafaxine hydrochloride tablets (10.28 mg/kg), the blood samples were collected in succession at different points in time. After 1-week washout period, Danzhi Xiaoyao pills (0.6g/kg) were given through oral administration to the six beagles every morning until the 7th day, venlafaxine hydrochloride tablets (10.28 mg/kg) were given after feeding Danzhi Xiaoyao pills (0.6g/kg) half an hour and blood samples were collected continuously at different points. All samples were analyzed by UPLC-MS/MS, and the main pharmacokinetic parameters of venlafaxine, ODV and NDV were computed by DAS 2.0. RESULTS: The Cmax of the venlafaxine group (control group) and the combination group (experimental group) were (2267.26±252.89) ng/mL and (1542.64±190.73) ng/mL, respectively. The AUC(0-∞) of the two groups were (13,934.79±3609.23) ng·h/mL and (8001.91±2167.58) ng·h/mL, respectively. The ODV Cmax of the two groups were (2253.80±215.81) ng/mL and (2721.37±118.20) ng/mL, and AUC(0-∞) were (13,974.99±2784.04) ng·h/mL and (17,539.44±1894.29) ng·h/mL, respectively. The NDV Cmax of the two groups were (50.98±5.76) ng/mL and (58.74±12.33) ng/mL, and AUC(0-∞) were (179.26±34.94) ng·h/mL and (220.68±51.41) ng·h/mL, respectively. After administration of Danzhi Xiaoyao pills, the Cmax and AUC(0-∞) of venlafaxine decreased significantly, indicating that the plasma exposure of venlafaxine decreased. The increase of Cmax and AUC(0-∞) of ODV and NDV indicated a rise in plasma exposure. CONCLUSION: Danzhi Xiaoyao pills can accelerate the metabolism of venlafaxine in beagles. In clinical, when venlafaxine was co-administrated with Danzhi Xiaoyao pills, dose adjustment of venlafaxine should be taken into account.

The virus-induced protein APOBEC3G inhibits anoikis by activation of Akt kinase in pancreatic cancer cells.

Pancreatic cancer is one of the more common cancers with a poor prognosis. Some varieties of cancer are related to virus infection. As a virus-induced protein, APOBEC3G (A3G) presents extensive anti-virus ability, but the role of A3G in pancreatic cancer was previously unknown. The expression of A3G in pancreatic cancer was examined using TaqMan real-time qPCR, immunohistochemical and immunofluorescent staining. Subsequently, the role of A3G in pancreatic cancer was evaluated in vivo using the tumor xenograft model. Anoikis was detected by colony formation assay and flow cytometry in vitro. The Akt kinase activity and target protein PTEN were examined by co-immunoprecipitation and immunoblot. The virus-induced protein A3G was significantly up-regulated in pancreatic cancer, and the up-regulation of A3G promoted xenograft tumor formation. A3G inactivated PTEN by binding to the C2 tensin-type and PDZ domains, thereby inducing anoikis resistance through Akt activation. Our results demonstrate that the up-regulation of A3G in pancreatic cancer cells induces anoikis resistance, and they provide novel insight into the mechanism by which A3G affects the malignant behavior of pancreatic cancer cells.

Including explicit water molecules as part of the protein structure in MM/PBSA calculations.

Water is the natural medium of molecules in the cell and plays an important role in protein structure, function and interaction with small molecule ligands. However, the widely used molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method for binding energy calculation does not explicitly take account of water molecules that mediate key protein-ligand interactions. We have developed a protocol to include water molecules that mediate ligand-protein interactions as part of the protein structure in calculation of MM/PBSA binding energies (a method we refer to as water-MM/PBSA) for a series of JNK3 kinase inhibitors. Improved correlation between water-MM/PBSA binding energies and experimental IC50 values was obtained compared to that obtained from classical MM/PBSA binding energy. This improved correlation was further validated using sets of neuraminidase and avidin inhibitors. The observed improvement, however, appears to be limited to systems in which there are water-mediated ligand-protein hydrogen bond interactions. We conclude that the water-MM/PBSA method performs better than classical MM/PBSA in predicting binding affinities when water molecules play a direct role in mediating ligand-protein hydrogen bond interactions.

Role of Helicobacter pylori virulence factor cytotoxin-associated gene A in gastric mucosa-associated lymphoid tissue lymphoma.

Helicobacter pylori (H. pylori) infection might initiate and contribute to the progression of lymphoma from gastric mucosa-associated lymphoid tissue (MALT). Increasing evidence shows that eradication of H. pylori with antibiotic therapy can lead to regression of gastric MALT lymphoma and can result in a 10-year sustained remission. The eradication of H. pylori is the standard care for patients with gastric MALT lymphoma. Cytotoxin-associated gene A (CagA) protein, one of the most extensively studied H. pylori virulence factors, is strongly associated with the gastric MALT lymphoma. CagA possesses polymorphisms according to its C-terminal structure and displays different functions among areas and races. After being translocated into B lymphocytes via type IV secretion system, CagA deregulates intracellular signaling pathways in both tyrosine phosphorylation-dependent and -independent manners and/or some other pathways, and thereby promotes lymphomagenesis. A variety of proteins including p53 and protein tyrosine phosphatases-2 are involved in the malignant transformation induced by CagA. Mucosal inflammation is the foundational mechanism underlying the occurrence and development of gastric MALT lymphoma.

ST13, a proliferation regulator, inhibits growth and migration of colorectal cancer cell lines.

BACKGROUND AND OBJECTIVE: ST13, is the gene encoding the HSP70 interacting protein (HIP). Previous research has shown that ST13 mRNA and protein levels are down-regulated in colorectal cancer (CRC) tissues compared with adjacent normal tissues. This study aims at the role of ST13 in the proliferation and migration of CRC cells. METHODS: The transcript level of ST13 in different CRC cell lines was evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). ST13-overexpressed and ST13-knockdown CRC cells were constructed respectively by lentiviral transduction, followed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, plate colony formation, cell-cycle analysis, and migration assays to evaluate the influence of ST13 on proliferation and migration in vitro. Moreover, a mouse xenograft study was performed to test in vivo tumorigenicity of ST13-knockdown CRC cells. RESULTS: Lentivirus-mediated overexpression of ST13 in CRC cells inhibited cell proliferation, colony formation, and cell migration in vitro. In contrast, down-regulation of ST13 by lentiviral-based short hairpin RNA (shRNA) interference in CRC cells significantly increased cell proliferation and cloning efficiency in vitro. In addition, down-regulation of ST13 expression significantly increased the tumorigenicity of CRC cells in vivo. CONCLUSIONS: ST13 gene is a proliferation regulator that inhibits tumor growth in CRC and may affect cell migration.

Proteomic analysis of primary colon cancer-associated fibroblasts using the SELDI-ProteinChip platform.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts. METHODS: Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform. RESULTS: Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin ß-4-like protein 3, and tight junction-associated protein 1. CONCLUSIONS: Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.

Inhibition of matrine against gastric cancer cell line MNK45 growth and its anti-tumor mechanism.

Anti-tumor activity and mechanism of matrine is evaluated and investigated. MTT assay showed that the matrine was able to inhibit gastric cancer cell line MNK45 in a dose-dependent manner. The concentration required for 50% inhibition (IC50) was found to be 540 µg/ml. This anti-tumor function was achieved through modulation of the NF-κB, XIAP, CIAP, and p-ERK proteins expression in cell line MNK45. By western blot analysis, we found that expression of NF-κB, XIAP, CIAP, and p-ERK proteins in cell line MNK45 would vary with varying concentration of matrine. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyzes are need. These results overall indicate that matrine can be used as an effective anti-tumor agent in therapy of gastric cancer.

Induced pluripotent stem cell-related genes influence biological behavior and 5-fluorouracil sensitivity of colorectal cancer cells.

OBJECTIVE: We aimed to perform a preliminary study of the association between induced pluripotent stem cell (iPS)-related genes and biological behavior of human colorectal cancer (CRC) cells, and the potential for developing anti-cancer drugs targeting these genes. METHODS: We used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the transcript levels of iPS-related genes NANOG, OCT4, SOX2, C-MYC and KLF4 in CRC cell lines and cancer stem cells (CSCs)-enriched tumor spheres. NANOG was knockdowned in CRC cell line SW620 by lentiviral transduction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, plate colony formation, and a mouse xenograft model were used to evaluate alterations in biological behavior in NANOG-knockdown SW620 cells. Also, mock-knockdown and NANOG-knockdown cells were treated with 5-fluorouracil (5-FU) and survival rate was measured by MTT assay to evaluate drug sensitivity. RESULTS: A significant difference in the transcript levels of iPS-related genes between tumor spheres and their parental bulky cells was observed. NANOG knockdown suppressed proliferation, colony formation, and in vivo tumorigenicity but increased the sensitivity to 5-FU of SW620 cells. 5-FU treatment greatly inhibited the expression of the major stemness-associated genes NANOG, OCT4, and SOX2. CONCLUSIONS: These results collectively suggest an overlap between iPS-related genes and CSCs in CRC. Quenching a certain gene NANOG may truncate the aggressiveness of CRC cells.

Oxaliplatin-incorporated micelles eliminate both cancer stem-like and bulk cell populations in colorectal cancer.

PURPOSE: The failure of cancer treatments is partly due to the enrichment of cancer stem-like cells (CSLCs) that are resistant to conventional chemotherapy. A novel micelle formulation of oxaliplatin (OXA) encapsulated in chitosan vesicle was developed. The authors postulate that micelle encapsulation of OXA would eliminate both CSLCs and bulk cancer cells in colorectal cancer (CRC). EXPERIMENTAL DESIGN: In this study, using stearic acid-g-chitosan oligosaccharide (CSO-SA) polymeric micelles as a drug-delivery system, OXA-loaded CSO-SA micelles (CSO-SA/OXA) were prepared. Intracellular uptake of CSO-SA/OXA micelles was assessed by confocal microscope. The effects of free OXA, the empty carrier, and CSO-SA/OXA micelles were tested using human CRC cell lines in vitro and in vivo. RESULTS: The micelles showed excellent internalization ability that increased OXA accumulation both in CRC cells and tissues. Furthermore, CSO-SA/OXA micelles could either increase the cytotoxicity of OXA against the bulk cancer cells or reverse chemoresistance of CSLC subpopulations in vitro. Intravenous administration of CSO-SA/OXA micelles effectively suppressed the tumor growth and reduced CD133+/CD24+ cell (putative CRC CSLC markers) compared with free OXA treatment, which caused CSLC enrichment in xenograft tumors (P < 0.05). CONCLUSION: The results of this study indicate that CSO-SA micelle as a drug-delivery carrier is effective for eradicating CSLCs and may act as a new option for CRC therapy.

Isolation and characterization of radiation-resistant lung cancer D6-R cell line.

OBJECTIVE: To isolate an isogenic radioresistant cancer cell line after fractioned X-ray radiation and characterize the resistant cells. METHODS: D6 cells were exposed to repeated X-ray irradiation, and after a total dose of 5200 cGy in 8 fractions, a radioresistant monoclone D6-R was obtained. The radiosensitivity and drug sensitivity of the novel radioresistant D6-R cells, together with their parent D6 cells, were measured using clonogenic assay and MTT assay respectively. Cell cycle distribution was analyzed by flow cytometry. Fluorescence microscopy and flow cytometry were applied for apoptosis detection. Comet assay was used for the detection of DNA damage and repair. RESULTS: D6-R cells showed higher and broader initial shoulder (D0=2.08 Gy, Dq=1.64 Gy, N=2.20) than the parent D6 cells (D0=1.84 Gy, Dq=0.34 Gy, N=1.20). They were 1.65-fold more radioresistant than D6 cells in terms of SF2 (63% vs 38%) and were more resistant to ADM (3.15-fold) and 5-FU (3.86-fold) as compared with the latter. It was found that D6-R cells had higher fractions of cells in S phase (53.4% vs 37.8%) and lower fractions of cells in G1 (44.1% vs 57.2%) and G2-M phase (2.5% vs 5%). There was no difference in radiation-induced apoptosis between D6-R and D6 cells. D6-R cells showed less initial DNA damage and increased capacity in DNA repair after irradiation, as compared with the parent cells. CONCLUSIONS: D6-R cells have been isolated by exposing the parental D6 cells to repeated irradiation. The difference in cell cycle pattern together with the induction and repair of DNA damage might, at least partially, explain the mechanism of the radioresistance.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.