L. coronopifolia against human breast carcinoma cell line (MDA-MB-321). Breast cancer is the most common cause of deaths in women. The search for traditionally used medicinal plants which can serve as non-toxic and affordable anticancer drugs is the need of the hour. This study aimed to investigate the anticancer potential of extracts of L. coronopifolia for 24 h. The cytotoxic response of different extracts was measured by MTT assay, neutral red uptake (NRU) assay and cellular morphological alterations under the microscope.The MDA-MB-231 cells were plated in 96 well plates and exposed to 10-1,000 µg/ml of L. coronopifolia exposed MDA-MB-231 cells. The results also exhibited morphological alterations in MDA-MB-231 cells exposed to various extracts. The cells treated with 250- 1000 µg/ml lost their original morphology and cell linkage as compared to control cells. A concentration-dependent decrease in the cell viability of MDA-MB-231 cells was observed after the exposure of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia. The cell viability was found to be 82%, 89% and 98% at 1000, 500 and 250 µg/ml, respectively in petroleum ether, 37%, 75% and 88% at 1,000, 500 and 250 µg/ml, respectively in ethyl acetate extract, 30%, 35% and 64% at 1,000, 500 and 250 µg/ml, respectively in chloroform extract and 44%, 65% and 82% at 1000, 500 and 250 µg/ml, respectively in ethanolic extract of L. coronopifolia against MDA-MB-321 cells. Further studies are required to know the mechanism(s) involved in the cell death.These preliminary results suggest the promising anticancer potential of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of  The cancer has been recognized as second foremost cause of death worldwide after cardiovascular diseases . Among women, breast cancer is the most common type of cancer and the major cause of death . In developing countries, breast cancer is prominent reason of death and in developed countries, breast cancer is second leading cause of death . In spite of new diagnosis techniques such as mammography and needle sampling, no significant change has been observed in the mortality rate since many decades. The increasing tumor heterogeneity, drug resistance and high cost of available therapeutic methodologies are present concern that are connected with effective treatment of breast cancer . Although, excessive progresses have been made to understand the pathophysiology of disease and the development towards conventional chemotherapy even though there is still need to explore effective drug to treat the breast cancer . Thus the novel, non-toxic, target oriented chemotherapeutic agents at affordable cost are yet to be achieved. L. augustifolia exhibited anti-inflammatory and analgesic activities . L. stoechas displayed hepato- and neuroprotective properties . L. pubescens is known for possessing a broad spectrum antimicrobial , anti-inflammatory and hepatoprotective activities . L. coronopifolia Poir, L. dentata and L. pubescens Decne are the three species found in Saudi Arabia. These plants have been used in traditional system of medicine. The infusion of flowers of L. dentata is used for urine retention and removal of stones from kidney and ureter. Similarly L. pubescens is known to relieve headache and cold . The literature survey also reveals L. coronopifolia possess antioxidant , antibacterial  and hepatoprotective potential . However, the anticancer properties of L. coronopifolia have not been reported so far. Hence, this study was designed to explore the anti-cancer effect of various solvent extracts of L. coronopifolia on human breast carcinoma cell line (MDA-MB-321).For centuries now, plants have been used in nutrition, cosmetics and medicine. Owing to the presence of various bioactive metabolites, medicinal plants exhibit various pharmacological activities viz. antifungal, antibacterial, antioxidant, spasmodic, anticancer etc. . Traditional medicinal plants have been proven to play an important role in the treatment of various cancer diseases, since ancient . Therapeutically significant bioactive compounds, such as Taxol, Vinblastine, Vinflunine, Vinorelbine, Camptothecin, Vincristine and Vindesine have been explored from plants and are established to treat the various cancer diseases . The genus Lavandula consists of 39 species of flowering plants in family Lamiaceae . The plants belonging to this genus are mainly are annuals, shrubs and herbaceous plants. Lavandula plants are known for their antioxidant, insecticidal, anti-inflammatory, sedative and spasmodic properties . Chemicals and consumablesCell culture medium (DMEM), Fetal bovine serum, and trypsin-EDTA (0.25%) were procured from Gibco, Invitrogen, USA. Cell culture plates and other plastic wares used in present study were purchased from Nunc. MTT, neutral red dye, other specified chemicals and solvents were obtained from Sigma. Plant material and extractionsLavandula coronopifolia plant were air dried and grounded to a coarse powder. For the extraction, powdered plant material was macerated in petroleum ether, ethyl acetate, chloroform, and ethanol successively. The procedure was repeated several times. At each stage the filtrate was collected and the solvent was then evaporated to obtain the extracts of respective solvents. The plant used in present investigation was collected from Shaza Mountains in Saudi Arabia. The plant was authenticated by a taxonomist and a voucher (# 15799) was submitted at the herbarium, Department of Pharmacognosy, College of Pharmacy, King Saud University. The aerial parts of Cell culture2 incubator at 37oC in high humid atmosphere. MDA-MB-231, a human breast cancer cell line was originally purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The cell line, MDA-MB-231 was grown in DMEM medium with 10% fetal bovine serum, 0.2% NaHCO3 and 1% antibiotic/antimycotic solution in a COExperimental designL. coronopifolia for 24 h. After the treatment, cytotoxicity assessment of L. coronopifolia extracts was done using MTT assay, neutral red uptake assay, and morphological assessments on MDA-MB-231 cell line. MDA-MB-231 cell line was exposed to 10 - 1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of Cytotoxicity Assessment of L. coronopifolia extracts (MTT assay)L. coronopifolia extracts was performed using MTT assay as described by Mossman et al., 1983. In brief, MDA-MB-231 cells  were seeded in 96 well culture plates and allowed to adhere in CO2 incubator for overnight. Then, MDA-MB-213 cells were treated with 10 - 1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After exposure, 10 μl/well MTT solution (5 mg/ml) was added and incubated further for 4 h. Supernatant was removed from the wells and DMSO (200 µl) was added in each well and mixed. The absorbance of plate was then read at 550 nm wave length.Cytotoxic effects of Cytotoxicity Assessment of L. coronopifolia extracts (NRU assay)L. coronopifolia extracts was also assessed by NRU assay as described by Borenfreund and Puerner, 1984. In brief, MDA-MB-231 cells  were seeded in 96 well culture plates and allowed to adhere in CO2 incubator for overnight. Then, cells were exposed to various concentrations  of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After the treatment, neutral red (50 μg/ml) was added in wells and further incubated for 3 h. Then, solution was rapidly washed with solution  and the dye was extracted in a solution of 1% acetic acid and 50% ethanol. The absorbance of plate was then read at 550 nm wave length.Cytotoxic effects of Morphological analysisLavandula coronopifolia for 24 h. After the treatment with Lavandula coronopifolia, cell images of MDA-MB-231 cells were grabbed under the phase contrast inverted microscope at 20× magnification. For the analysis of morphological assessment, the MDA-MB-231 cell line was exposed to 10 - 1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of Statistical analysisThe p values <0.05 were taken as statistically significant between control and treated groups using One-way ANOVA. The results are expresses as Mean + SD from three independent experiments.Cytotoxicity Assessment of L. coronopifolia extracts (MTT assay)L. coronopifolia against MDA-MB-231 cell line obtained by MTT assay are presented in L. coronopifolia for 24 h exhibited significant decrease in the cell viability of MDA-MB-231 cell line in a concentration dependent manner. The percent cell viability was found as 82%, 89% and 98% at 1,000, 500 and 250 µg/ml, respectively in petroleum ether extract L. coronopifolia obtained by NRU assay. Like MTT assay, similar kind of cytotoxic effects in MDA-MB-231 cells treated with L. coronopifolia extracts were also observed by NRU assay. A concentration dependent cytotoxic effects was observed in MDA-MB-231 cells exposed to 10-1,000 µg/ml for 24 h. The percent cell viability recorded by NRU assay was 83%, 90% and 97% at 1,000, 500 and 250 µg/ml, respectively in petroleum ether extract  cell line. The MDA-MB-231 cell line in this study was chosen, because it has been proven as a good in vitro model system to investigate the cytotoxicity and anticancer potential of various plant extracts against breast cancer . Traditionally medicinal plants have been proven to play an important role in the treatment of various cancer diseases . Numerous researches have been shown beneficial effects of plant extracts against various cancer cell lines . It is well documented that plants and their components could act as cytotoxic/antiproliferative effects and induced apoptosis in various cancer cell lines . These days, the research on natural/plant phytochemicals are increasing actively because of their potential health benefits and less side effects. In this study, we have chosen Lavandula coronopifolia plant because it is reported that L. coronopifolia possess antioxidant , antibacterial  and hepatoprotective potential against ethanol induced oxidative stress and cytotoxicity . But anticancer properties of Lavandula coronopifolia have not been reported against human breast carcinoma cells. The cytotoxic effects of potential of different extracts of L. coronopifolia has been assessed using various parameters, such as MTT assay, NRU assay and morphological alterations in MDA-MB-231 cell line treated with different concentrations for 24 h. The MTT and NRU assays showed that petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia decreases the cell viability of MDA-MB-231 cell line in a concentration dependent manner. Breast cancer is one of the vigorous problems and major cause of mortality in female globally. Therefore, the aim of this study was to investigate anticancer potential of Lavandula coronopifolia did not show significant cytotoxicity to MDA-MB-231 cells. The concentrations range selected in study was chosen from previously reported literature, since exposure of different natural products have been studied by different investigators under in vitro conditions . The results of the present study are in accordance to previous report on cytotoxic potential of different plant extracts . The ethanolic extract of Nigella sativa plant have also been reported to possess cytotoxic effects against P388, HepG2, Molt4 and Lewis lung carcinoma cell lines . The ethyl acetate extract of Bismillah leaf  induced cytotoxicity and morphological alterations have also been reported against human glioblastoma cell line (U-87) . In other study, the cytotoxic/antiprolifertaive potential of ethyl acetate extract against HT-29 human colon cancer and MCF-7 human breast cancer cells have also been reported . The anticancer potential of chloroform extract and sub-fractions of Nepeta deflersiana against MCF-7 human breast cancer and A549 human lung carcinoma cell lines have been studied . Recently, Usmani et al., (2018) have also reported the antiproliferative and cellular oxidative stress induced by Cordia dichotoma (Linn.) extract and its fractions on human cervix epitheloid (HeLa) and human lung (A549) carcinoma cells. The anticancer/cytotoxic effects of L. coronopifolia extracts in this investigation suggest that the cytotoxic response of different extracts obtained could be due to the presence of active components, which is showing the anticancer activity of L. coronopifolia extracts against human breast cancer cells MDA-MB-231. The chloroform extract was found to be more cytotoxic towards MDA-MB231 followed by ethyl acetate extract and ethanolic extract. Petroleum ether extract was found to be less cytotoxic as compared to other extracts. The cytotoxicity of ethyl ether, chloroform and ethanolic extracts were found at 250 µg/ml and above concentrations of Lavandula coronopifolia, whereas, 100 µg/ml and lower concentrations of L. coronopifolia decreased the cell viability and altered the cell morphology of human breast cancer cell line (MDA-MB-321). The results also showed that chloroform extract was found to be more cytotoxic towards MDA-MB-231 followed by ethyl acetate extract and ethanolic extract. Petroleum ether extract was found to be less cytotoxic as compared to other extracts. Further, more studies are required to know the mechanism(s) of cell death involved in this process. In conclusion, the results from present investigation demonstrated that petroleum ether, ethyl acetate, chloroform, and ethanol extracts of


EWSR1-FLI1, PAX3-FOXO1 and SS18-SSX, respectively. Finally, animal experiments confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for treating sarcomas, especially those harboring chromosomal translocation.Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine, with the development of novel drugs being of keen interest. A pan-PI3K inhibitor, ZSTK474, has been evaluated in clinical trials against a range of advanced solid tumors, with clinical benefit shown in sarcoma patients. In the present study, we developed a panel of 14 human sarcoma cell lines and investigated the antitumor effect of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment. ZSTK474 exhibited a similar antiproliferative profile to other PI3K inhibitors but was clearly different from the other drugs examined. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all cell lines examined, showing proof-of-concept of PI3K inhibition. In addition, ZSTK474 induced apoptosis selectively in Ewing's sarcoma (RD-ES and A673), alveolar rhabdomyosarcoma (SJCRH30) and synovial sarcoma  cell lines, all of which harbor chromosomal translocation and resulting oncogenic fusion genes,  EWSR1  gene and one of the ETS transcriptional factor genes (FLI1 or ERG) [SS18  gene and one of the SSX  genes  [Sarcomas are rare malignant tumors of mesenchymal origin, such as muscle, fat, bone and other connective tissues, with > 50 different subtypes reported to date . It is e or ERG)  and synoor SSX4) –9. Theseor SSX4) –12. Anotor SSX4) .Although several animal models and patient-derived xenograft models have been established to understand the molecular mechanism underlying sarcoma development, few therapeutics options are available thus far due to its complex character . The priin vitro tool to identify novel antitumor agents and predict modes of action, as well as to identify predictive biomarkers relating to antitumor efficacy. “Fingerprints” are defined as the patterns of differential drug efficacy across the panel of cell lines, and have been found to be reflective of mode of action. Indeed, the mode of action of novel anticancer compounds could be predicted by correlation analysis of its fingerprint to those of reference compounds via a bioinformatic approach called COMPARE [in vitro and in vivo [We previously established a panel of 39 human cancer cell lines (JFCR39) composed of various carcinoma types –24. The  COMPARE . Using t COMPARE . This co in vivo –30. Clin in vivo . Interesvia the use of a cell line panel approach, akin to JFCR39. We collected 14 commercially-available sarcoma cell lines from various origins and established a sarcoma panel. A total of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment were examined with respect to their antitumor profiles across the panel of sarcoma cell lines in terms of effects on tumor growth, PI3K-downstream signaling pathway alterations and apoptosis induction in vitro and in vivo. These profiles were then compared to genetic backgrounds of sarcoma cells in an attempt to identify candidate predictive markers for the antitumor efficacy of ZSTK474.In the present study, we characterized the antitumor profile of ZSTK474 in sarcoma cells TP53, which was the most frequently mutated gene among the genes examined. In addition, gain of function mutations, which gave new activity or enhancement of its activity to the proteins, were commonly found in KIT , BRAF  and NRAS  genes. In contrast, none of the cell lines in this panel harbored known gain of function mutations in the PIK3CA gene at the hotspot residues . Missense mutations were not observed in the PTEN gene in these cell lines, while intronic deletions were observed in the HT-1080, RD and RD-ES cell lines.We first examined the genetic background of 14 cell lines within the sarcoma panel by amplicon sequencing. The origin and mutation status of each of the cell lines is shown in Table We next examined the expression and the activation status of signaling proteins involved in PI3K-AKT-mTOR and RAS-MEK-ERK pathways by immunoblot analysis, as shown in Figure NRAS, KRAS or BRAF genes expressed phosphorylated MEK and ERK proteins at a significantly higher level than wild-type cell lines  concentrations also calculated , docetaxel (r = –0.63) or gemcitabine (r = –0.57). In addition, we found a significant positive correlation between phosphorylated IGF1R and the IGF1R-TKI, linsitinib (r = 0.63) or the PI3K-alpha specific inhibitor alpelisib (r = 0.55), and between phosphorylated MEK and sensitivity to selumetinib (r = 0.66) or vemurafenib (r = 0.55).We secondly examined the correlation between drug efficacy and activation status; in vitro, we examined the phosphorylation status of PI3K-downstream signaling molecules after treatment with ZSTK474. As shown in Figure To confirm proof of concept of PI3K inhibition in vitro, we next exploited sarcoma xenograft models to determine antitumor activity of ZSTK474 in vivo. Of the 14 sarcoma cell lines, three cell lines  were subcutaneously transplantable and exhibited reproducible tumor growth in nude mice (data not shown). Therefore, we examined the effect of ZSTK474 on growth of xenografted tumors derived from these cell lines, and compared it with that affected by doxorubicin and pazopanib. As shown in Figure via inhibiting the PI3K signaling pathway in vivo, as well as in vitro.Given that PI3K inhibitors showed antiproliferative activity in sarcoma cell lines via G1 arrest of the cell cycle, but not by a cytotoxic effect via apoptosis, using carcinoma cells from various origins both in vivo and in vitro [50 for an extended period  [The most common chromosomal translocation found in Ewing's sarcoma, t, generates an aberrant transcription factor (BEZ235) , suggestEWSR1-FLI1-negative osteosarcoma cell line, HOS. This result suggested that downregulation of Id2 protein expression observed in Ewing's sarcoma cell lines upon treatment with ZSTK474 could be mediated via transcriptional inactivation of EWSR1-FLI1 from the Id2 promoter.Then, we next examined the effect of ZSTK474 on transcriptional activity of EWSR1-FLI1 by reporter assays. As shown in Figure in vivo. A673 cells were subcutaneously transplanted in nude mice, with the rapid growth of tumors subsequently observed. ZSTK474 administration for two weeks exerted a robust antitumor activity against A673-derived tumors -SSX2 fusion gene, underwent apoptosis upon treatment with ZSTK474, as determined by emergence of cleaved PARP  compared to the MEK inhibitor selumetinib (~1000-fold range). These results suggested that ZSTK474 effectively suppressed tumor growth in sarcoma cell lines irrespective of their subtype. Moreover, ZSTK474 downregulated phosphorylation of PI3K-downstream signaling factors including AKT and ribosomal S6 protein in all of the sarcoma cell line tested, suggesting proof of concept of PI3K inhibition.In the present study, we characterized the antitumor profile of PI3K inhibitors, in particular ZSTK474 which we developed, against sarcomas in preclinical models by exploiting a panel of sarcoma cell lines derived from various origin; in this respect, effects on tumor growth, PI3K-downstream signaling pathway and apoptosis induction KRAS or BRAF genes and overexpression of IGF1R were found to be negative predictors whereas high expression of phosphorylated AKT was observed to be a positive predictor for ZSTK474 efficacy [KRAS/NRAS/BRAF nor overexpression of IGFR/phosphorylated AKT displayed any significant correlation with ZSTK474 efficacy in the sarcoma panel assessed here. HT-1080, one of the less effective cell lines examined, harbored gain of function mutation of NRAS, suggesting that NRAS mutation could be involved ZSTK474 inefficacy; however, RD, another NRAS mutant cell line, responded to ZSTK474 at a moderate level. Further studies are needed to clarify whether NRAS mutation predicts ZSTK474 inefficacy. In addition, we could not determine whether PIK3CA mutation could predict the efficacy of ZSTK474, since none of the 14 sarcoma cell lines examined harbored a gain of function mutation of PIK3CA despite gain of function mutations in this genes being commonly found in in 18–25% of patients with liposarcomas [In our previous study using the JFCR39 carcinoma cell line panel, gain of function mutations of the efficacy ; howeversarcomas , 42. LosEWSR1-FLI1 in Ewing's sarcoma RD-ES cell line and PAX3-FOXO1 in alveolar rhabdomyosarcoma SJCRH30 cell line, respectively) [SS18-SSX2 fusion gene [In contrast to growth inhibitory activity, apoptosis induction was selectively observed following ZSTK474 treatment. As summarized in Table ctively) , 44. We ion gene , and fouvia a reporter assay, and suppressed expression of the Id2 protein in both of the Ewing's sarcoma cell lines examined. Actually, a previous study demonstrated that the EWSR1-FLI1 fusion could transform cells [SS18-SSX fusion genes are capable of transforming cells and knockdown of SS18-SSX expression can greatly reduce viability of synovial sarcoma cells, as determined by colony formation assays [via PI3K/AKT inhibition. However, the causality between these events observed in chromosomal translocation positive sarcoma cells and the induction of apoptosis were unclear in the context of this study. In fact, we detected neither common nor specific changes in the expression levels of Bcl-2 and IAP (inhibitor of apoptosis) family proteins in these cells upon treatment with ZSTK474 (data not shown). Further studies are needed to understand the precise mechanism of apoptosis induction in such types of sarcoma cells.Here, we demonstrated that ZSTK474 partially downregulated transcriptional activity of the EWSR1-FLI1 transcription factor from the Id2 promoter rm cells , and down assays . Interesn assays , 50. Sinn assays , 52, seln assays , and thuin vivo antitumor effect of ZSTK474 using various sarcoma cell lines subcutaneously xenografted in nude mice. ZSTK474 exerted a significant in vivo antitumor effect against all of sarcoma cell lines examined, comparable to the existing antitumor drugs doxorubicin and pazopanib. Of note, in vivo effect was most obvious in the three chromosomal translocation-positive sarcoma cell lines that were characterized with apoptosis induction . Doxorubicin was purchased from Cayman Chemical Company  and dissolved with saline . Pazopanib was purchased from Cellagen Technology , dissolved with DMSO for in vitro studies, and suspended with 5% hydroxypropylmethyl cellulose (HPMC) for in vivo studies.Buparlisib (BKM-120) and everolimus (RAD-001) were purchased from AdooQ BioScience . Sorafenib, doxorubicin and gemcitabine were purchased from Cayman Chemical Company . Cisplatin, pictilisib (GDC-0941) and pazopanib were purchased from Cellagen Technology . Docetaxel and selumetinib (AZD6244) were purchased from Focus Biomolecules . Carboplatin and ifosfamide were purchased from LKT Laboratories, Inc. . Alpelisib (BYL-719), vemurafenib, imatinib, sunitinib and gefitinib were purchased from Active Biochemicals Co., Ltd. , Axon Medchem BV. , BioVision Inc. , R&D Systems, Inc.  and Synkinase Pty Ltd. , respectively. ZSTK474, ZSTK534, ZSTK778, ZSTK1741 and ZSTK2209 were kindly gifted from Zenyaku Kogyo Co., Ltd. For 2. A673 was also purchased from American Type Culture Collection  and cultured in the same condition mentioned above. SYO-1 cell line provided by Dr. Akira Kawai  and Aska-SS and Yamato-SS cell lines provided by Dr. Norifumi Naka  were cultured in the same condition.Cell lines used in the sarcoma cell line panel assay are listed in Table via the TaKaRa Amplicon sequencing service by using the Ion Ampliseq Cancer Hotspot Panel v2, which includes primer sets to amplify 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes using human hg19 reference genome as described in the manufacturer's web site .Genomic DNA was isolated from each cell line by DNeasy Blood & Tissue Kit according to the manufacturer's protocols . Sequencing library construction and Ion torrent amplicon sequencing were performed Inhibitory effects of each agent against the sarcoma cell lines was assessed by using the sulforhodamine B (SRB) assay . PercentPercentage growth (%) = 100 × (T–I)/(C–I)T: absorbance for the test well after incubation with drug for 48 hI: absorbance for the test and control well at starting point (addition of drugs),C: absorbance for the control well (C) after incubation with vehicle for 48 h50 was calculated as described previously . All drugs were tested in duplicate. The GI50 values were log transformed and were used for all analyses. Cluster analysis and visualization of heatmaps was performed using the Cluster 3.0 and TreeView software .The drug concentration of the GIvia fluorescence microscopy. The number of the cells with nuclear condensation and DNA fragmentation was counted and the percentage of apoptotic cells relative to total cells was calculated. At least 100 cells were counted in each group.Apoptosis was detected by Hoechst 33342 staining. Briefly, after incubation with ZSTK474 for 48 h, the cells were trypsinized, collected and fixed in ice cold 1% glutarardehyde-PBS (–) at 4° C for 24 h. After washing with PBS (–), the fixed cells were stained with 167 μM Hoechst 33342 , and observed ad libitium. Animal studies were performed in accordance with the procedures approved by the animal use and care committee of the Japanese Foundation for Cancer Research.Female nude mice with BALB/c genetic backgrounds  were purchased from Charles River Japan, Inc. , maintained under specific pathogen-free conditions in a 12:12 h light-dark cycle, and provided with sterile food and water Ectopic sarcoma xenograft models were previously established elsewhere by subcutaneous injection of SJCRH30 , SK-UT-12)/2,TW =  = 100 × average TW of drug-treated group/average TW of control group.3VO4, 5 mM EDTA, aprotinin at 100 Kal U/mL, 1 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS). Tumor samples were resected from mice 4 h after the final drug administration, minced with scissors and homogenized by a glass homogenizer with a Teflon pestle in the same buffer described above. The protein concentration of each sample was measured by a BCA Protein Assay Kit . Then, 10 μg of lysate from each cell line or tissue was separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the membrane was incubated with a primary antibody. Targeted antigens were detected by incubating with anti-phospho AKT (Ser473) antibody, anti-phospho AKT (Thr308) antibody, anti-pan AKT antibody, anti-phospho S6 antibody, anti-S6 antibody, anti-phospho IGF-1R β (Tyr1135/1136) antibody, anti-phospho MEK 1/2 (Ser217/221) antibody, anti-phospho ERK 1/2 (Thr203/Tyr204) antibody, anti-phospho 4E-BP1 (Thr37/46), anti-AKT antibody, anti-S6 antibody, anti-cleaved PARP at Asp214 antibody, anti-PTEN antibody, anti-Id2 antibody, anti-GAPDH antibody , anti-phospho IGF-1R antibody  or anti-α tubulin antibody , followed by fluorescently tagged secondary antibodies . Visualization of bound antibody was carried out using fluorescently tagged secondary antibodies  and the signals were detected and digitized by an Odyssey CLx Imaging System . An anti-α tubulin antibody  was used to provide data for a loading control.Cells were washed with ice cold PBS (–) twice and lysed with 10 mM Tris-HCl buffer at pH 7.4 containing 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 50 mM Na6 of cells (RD-ES and A673) or 2.5 × 105 cells (HOS) were seeded into 6 well culture plates and cultured for 24 h. Each cell was co-transfected with 1 μg of pGL3-Id2-2755 and 1 μg of Renilla (Renilla reniformis) luciferase internal control plasmid, pGL3-CMV  via the use of Lipofectamine 2000 . The culture medium was changed after 6 h of transfection, and cells were passaged to a 96 well plate and treated with ZSTK474 at final concentrations of 0.3 μM, 1 μM, 3 μM or 10 μM. Luciferase activity was detected using the Dual Glo Luciferase Assay System . The percentage of luciferase activity was calculated by the luciferase activity of firefly  luciferase derived from pGL3-Id2-2755 divided by the Renilla luciferase from pGL3-CMV. pGL3-Id2-2755 was kindly gifted by Dr. Tokino [1 × 10. Tokino .2O2 in PBS (–) and blocked with 10% normal goat serum. Following this, they were incubated with anti-p-S6 antibody, anti-cleaved PARP antibody  or anti-CD31 antibody . The bound antibodies were visualized by using EnVision+System HRP labeled polymer antibody and DAB kit , and the counterstaining was performed with hematoxylin. The number of nucleus of CD31-positive cell was counted as vascular endothelial cells.Tumors were resected from mice, fixed with neutral formalin and embedded in paraffin. Four μm sections were cut and deparaffinized in xylene, followed by rehydration through a graded ethanol series (100% to 50%) to PBS (–). After incubation with Dako Real Target Retrieval Solution, pH 6  in boiling water bath for 40 min, tumor sections were treated with 3% Hin vivo studies, n = 6). Statistical significance of antitumor efficacy was assessed by a Mann–Whitney U test or Student's t test or Welch t test for comparison of two samples on days 10 (MES-SA and MES-SA/Dx5) and days 14 . The statistical significance of observed anti-angiogenic effects was assessed by a Student's t test for comparison of two samples. The statistical significance of aberrant gene status and protein expression levels was determined by either a Mann–Whitney U test or Welch t test. The statistical significance of luciferase activity was assessed by a Student's t test. In all analyses, P < 0.05 was considered statistically significant.Data was expressed as mean ± SEM (


The HER2-enriched (HER2-E) subtype within HER2-positive (HER2+) breast cancer is highly addicted to the HER2 pathway. However, ∼20–60% of HER2+/HER2-E tumors do not achieve a complete response following anti-HER2 therapies. Here we evaluate gene expression data before, during and after neoadjuvant treatment with lapatinib and trastuzumab in HER2+/HER2-E tumors of the PAMELA trial and breast cancer cell lines. Our results reveal that dual HER2 blockade in HER2-E disease induces a low-proliferative Luminal A phenotype both in patient’s tumors and in vitro models. These biological changes are more evident in hormone receptor-positive (HR+) disease compared to HR-negative disease. Interestingly, increasing the luminal phenotype with anti-HER2 therapy increased sensitivity to CDK4/6 inhibition. Finally, discontinuation of HER2-targeted therapy in vitro, or acquired resistance to anti-HER2 therapy, leads to restoration of the original HER2-E phenotype. Our findings support the use of maintenance anti-HER2 therapy and the therapeutic exploitation of subtype switching with CDK4/6 inhibition. HER2-enriched breast cancers within the HER2-positive subtype are addicted to the HER2 pathway. Here, the authors analyse gene expression before, during, and after treatment with anti-HER2-based therapies in the phase II PAMELA clinical trial, finding phenotypic changes induced by treatment. Among them, the HER2-E is characterized by high expression of growth factor receptor-related genes  and cell cycle-related genes, low expression of estrogen-related genes such as ESR1 and PGR, and low expression of basal-related genes6. Although the HER2-E subtype is related to HER2+ disease as defined by immunohistochemistry, important discrepancies exist and HER2-E subtype represents ∼60%, ∼80% and ∼40% of HER2+, HER2+/hormone receptor-negative (HER2+/HR-negative) and HER2+/HR+ tumors, respectively7.Breast cancer consists of four molecular intrinsic subtypes  and a normal-like group20. However, not all HER2-E tumors achieve a complete response following anti-HER2-based therapies. For example, the pathological complete response (pCR) rates in HER2-E subtype following anti-HER2-based neoadjuvant therapy, with or without chemotherapy, are 40–80%21. At the same time, HER2-E tumors that do not achieve a pCR have a poor survival outcome22. Thus, there is a need to better understand the biology associated with incomplete response to HER2-targeted therapy in HER2-E disease. Here, we study patient’s tumor samples and preclinical models before, during and after anti-HER2 therapy to shed light on this clinical observation.The clinical value of the HER2-E subtype in HER2+ breast cancer is starting to be elucidated. From a therapeutic perspective, the HER2-E subtype has shown, across 14 clinical trials and ∼2,000 patients, higher sensitivity to anti-HER2-based therapies than the non-HER2-E subtypes7. The expression of the 50 PAM50 genes and 6 signatures  was explored at both time-points  and HR-negative (n = 61) disease, respectively  < 0.001) . Of note, normal tissue contamination at day 14 did not differ between PAM50 Luminal A tumor samples compared to PAM50 non-Luminal A tumor samples   with HER2+/HR+/HER2-E tumors (n = 38). The analysis revealed a strong correlation  between both gene lists, suggesting that anti-HER2 therapies have a higher impact on molecular tumor changes at day 14 than endocrine therapy  in the PAMELA phase II clinical trialnts Fig. . Among t23, where patients were treated for 2 weeks with either lapatinib, trastuzumab or the combination. Among the 56 genes/signatures, 85.7% were found differentially expressed (Supplementary Table p < 0.001)  or the lack of endocrine therapy.Next, we assessed the expression of the 56 genes/signatures in paired tumor samples from 36 patients with HER2+/HER2-E disease recruited in the LPT109096 phase II clinical trialPIK3CA that might confer resistance to anti-HER2 therapy24, or MCF7, which does not overexpress HER2  and 1 HER2-negative cell line . PAM50 subtyping of the 3 cell lines revealed that BT474, SKBR3 and HCC1954 are HER2-E and MCF7 is Luminal B  and a TKI (given at the IC50 for each cell line). Regarding individual PAM50 genes and signatures, 100% and 92.8% were found differentially expressed in BT474 and SKBR3, respectively (Supplementary Table p < 0.001) and the gene expression profile of each cell line was moderately correlated  with the gene expression profiles obtained from PAMELA and LPT109096  and the proliferation signature . Finally, BT474 cell line was identified by PAM50 as HER2-E as the parental cell line once anti-HER2 therapy was withdrawn  cell line and a BT474-derived tucatinib and trastuzumab resistant (BT474-TuRTR) cell line. Cell viability assays demonstrated that BT474-LRTR and BT474-TuRTR cells were resistant to combinations of lapatinib plus trastuzumab, or tucatinib plus trastuzumab, as opposed to parental BT474 , with FGFR4 being the most upregulated gene .To understand the changes in molecular phenotypes induced by chronic HER2 inhibition, we established a BT474-derived lapatinib and trastuzumab resistant  and leads to anti-HER2 resistance. For example, HER2-E tumors that became Luminal A at day 14 in the PAMELA trial32. Interestingly, a re-bound effect after stopping treatment has also been seen with other therapies such as CDK4/6 inhibitors28. Second, subtype switching from HER2-E to Luminal A might open an opportunity to treat the acquired phenotype with drugs that are known to be active in Luminal A, such as endocrine therapy and/or CDK4/6 inhibitors. For example, the PATRICIA phase II trial29 in advanced HER2+/HR+ disease revealed that the Luminal A tumors had improved progression-free survival when treated with palbociclib and trastuzumab as compared to HER2-E tumors. It is important to note that patients in the PATRICIA trial had been previously treated with a mean of 3 prior lines of anti-HER2-based therapy. Thus, HER2-E tumors at this stage are likely to have high anti-HER2 resistance and switching to a Luminal A phenotype is unlikely. Although this is a hypothesis and needs further validation, our preclinical data using HER2+ cell lines with a complete resistance to anti-HER2 treatment supports this observation.Our study highlights two additional aspects. First, the subtype switch seems reversible upon stopping anti-HER2 therapy. This could explain why maintenance therapy with anti-HER2 therapy both in the adjuvant and metastatic settings are beneficial to patients, especially in high-risk patients33, this provides a rationale for the concomitant HER2 and CDK4/6 inhibition in patients. Indeed, combinations of anti-HER2 therapies with CDK4/6 inhibitor and endocrine therapy are being investigated in 2 phase III trials for advanced HER2+/HR+ disease . Both trials differ in the population being evaluated. PATINA focuses on maintenance therapy in HER2+ tumors that are responding to anti-HER2 therapy, whereas monarcHER35 focuses on tumors that have progressed to at least 2 prior lines of anti-HER2 therapy. Concordant with our hypothesis, the monarcHER main results presented at ESMO 201936 revealed that abemaciclib + fulvestrant + trastuzumab was superior to chemotherapy + trastuzumab  most likely due to the luminal tumors deriving larger benefit form CDK4/6 inhibition and endocrine therapy compared to chemotherapy.The HER2 signaling pathway directly regulates the expression of Cyclin D1, affecting its interaction with CDK437, we have not explored the molecular effects in vitro of dual HER2 blockade with trastuzumab and pertuzumab, as our in vitro models cannot recapitulate antibody-dependent cellular cytotoxicity. Second, although our preclinical data suggest that a switch to Luminal A could be leveraged with CDK4/6 inhibition, we do not have clinical evidence that patients with HER2-E tumors that become Luminal A upon anti-HER2 therapy benefit from CDK4/6 inhibitors. Further clinical studies are needed to address this preclinical hypothesis.Our study has  two limitations worth noting. First, although a switch from HER2-E to Luminal A has been described after combinations of trastuzumab, pertuzumab and chemotherapy in clinical samplesIn conclusion, our findings support the use of maintenance anti-HER2 treatment in HER2+ breast cancer sensitive to anti-HER2 therapies, and warrant further research into exploiting molecular subtypes changes  to improve patient outcomes.7. This study is registered with ClinicalTrials.gov, number NCT01973660, and it is completed. The study protocol was approved by independent ethics committees at each center. In this trial, 151 patients with early HER2+ breast cancer were treated with neoadjuvant lapatinib (1000 mg daily) and trastuzumab (8 mg kg−1 i.v. loading dose followed by 6 mg kg−1) for 18 weeks. Patients with HR+ breast cancer received letrozole or tamoxifen according to menopausal status. Tumor samples were collected at baseline, day 14 and surgery and subsequently formalin-fixed paraffin-embedded (FFPE). As previously reported7, Ki67 was evaluated by IHC on FFPE tissue sections from core biopsies at baseline and at day 14 of treatment. These data are provided as a Source Data file.The main results of the PAMELA neoadjuvant phase II study have been previously reported23. This study is registered with ClinicalTrials.gov, number NCT00524303. In this trial, 100 patients with early HER2+ breast cancer were randomized to lapatinib, trastuzumab or both. Patients received 2 weeks of treatment without chemotherapy, and after a tumor biopsy, they received the same anti-HER2 treatment in combination with chemotherapy consisting of 5FU 500 mg m−2 + epirubicin 75 mg m−2 + cyclophosphamide 500 mg m−2 i.v. every 21 days (FEC75) for four cycles followed by weekly paclitaxel 80 mg m−2 for 12 weeks. Trastuzumab was administered every week (4 mg kg−1 i.v. loading dose followed by 2 mg kg−1). Lapatinib was administered every day . Tumor samples were collected at baseline and day 14 and subsequently FFPE according to protocol.The main results of the LPT109096 neoadjuvant phase II study have been previously reportedRTR) cell line and a BT474-derived tucatinib and trastuzumab resistant (BT474-TuRTR) cell line were established by treating BT474 with the corresponding TKI plus trastuzumab for 6 months, starting at low concentrations and increasing them at each passage. BT474-LRTR are maintained with 2 nM lapatinib + 2 µg ml−1 trastuzumab, while BT474-TuRTR are maintained with 2 nM tucatinib + 2 µg−1 ml trastuzumab.The breast cancer cell lines BT474, SKBR3 and MCF7 were purchased from the American Type Culture Collection . All cell lines were maintained as recommended by the suppliers. All cell lines were authenticated using Human 9-Marker STR Profile and Interspecies Contamination Test by IDEXX BioAnalytics. Lapatinib, neratinib, tucatinib and palbociclib were purchased from Selleckchem . Trastuzumab was obtained from Roche Farma . A BT474-derived lapatinib and trastuzumab resistant  from the PAMELA trial and 36 pairs of FFPE tumor samples (baseline and day 14) from the LPT109096 study using the High Pure FFPET RNA isolation kit  following manufacturer’s protocol. At least 1–5 10 μm FFPE slides were used for each tumor specimen, and macrodissection was performed to avoid contamination with normal breast tissue if needed. The RNeasy Mini Kit  was used to extract RNA from cell lines plated in triplicates in 6-well plates at a density of 150,000 cells per well. RNA was quantified at the NanoDrop spectrophotometer .RNA samples of the PAMELA trial from baseline and day 14 of treatment were previously extracted as reportedn = 101) and day 14 of treatment (n = 96) were previously analyzed7. Here, the nCounter platform  analyzed RNA samples from 57 residual tumors from the PAMELA trial, RNA from 36 paired tumors from the LPT109096 study, and RNA from cell lines treated with anti-HER2 and anti-CDK4/6 therapies. A minimum of 100 ng of total RNA was used to measure the expression of 50 genes of the PAM50 intrinsic subtype predictor assay and 5 housekeeping genes . Expression counts were then normalized using the nSolver 4.0 software and custom scripts in R 3.4.3. For each sample we calculated the PAM50 signature scores  and the proliferation signature score38. These data are provided as a Source Data file.RNA samples of the PAMELA trial from HER2-E tumors at baseline (−1 trastuzumab. Cell viability was determined 72 h after treatment using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)  following the manufacturer’s instructions, and quantified using the Gen5 Microplate Reader and Imager Software . For anti-CDK4/6 treatments, cells were plated in triplicate at 4000 cells per well in 96-well plates and treated at low doses of TKI or DMSO for 24 h. Cells were then treated with increasing concentrations palbociclib in combination with low doses of TKI or DMSO. At 72 h, cells were labeled with the DNA stain Hoechst 33342  and fluorescence was subsequently determined at the Gen5 Microplate Reader and Imager Software. GraphPad Prism was used for statistics. These data are provided as a Source Data file.Cells were plated in triplicate at 4000 cells per well in 96-well plates. The day after seeding, cells were treated with increasing concentrations of TKI  and 10 µg ml3O4V, 1 µM benzamidine, 1 µg ml−1 aprotinin, 1 µM leupeptin, 1 µM DTT. Total protein extracts were quantified using the DC Protein Assay . 25 μg of proteins were separated in reducing conditions (2.5% β-mercaptoethanol) by SDS–PAGE and transferred to nitrocellulose membranes  for further processing, following standard Western blotting procedures. Primary antibodies used in this study were: HER2 (D8F12), Phospho-HER2 (Tyr1221/1222) (6B12), AKT, Phospho-Akt (Ser473) (D9E), Phospho-RB (Ser807/811) (D20B12), Cyclin D1 (92G2) and anti-GAPDH (14C10) rabbit antibodies from Cell Signaling Technologies . The secondary fluorescent antibody used was the IRDye 800CW Donkey anti-Rabbit IgG . Fluorescent signal was acquired by the Odyssey Imaging System . Uncropped blots are provided as a Source Data file.Cells were plated in triplicate at 150,000 cells per well in 6-well plates and treated with TKI and trastuzumab. Cell lysates were collected after 24 h of treatment with Pierce RIPA buffer  supplemented with protease inhibitors: 5 µM NaF, 1 µM PMSF, 1 µM Na−1 trastuzumab and TKI  or with DMSO control. Propidium Iodide (PI) was used to stain total DNA. Data acquisition was performed at the Cytometry and cell sorting core facilities of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) using a BD FacsDiva analyzer. GraphPad Prism was used for statistics. These data are provided as a Source Data file.BC cell lines were fixed in 70% cold ethanol after 72 h of treatment with combinations of 10 µg ml−1 trastuzumab and TKI  or with DMSO control. After 10 days, cells were fixed with cold methanol and stained with 0.5% crystal violet solution.Breast cancer cell lines were plated at low density in 6-well plates and treated with combinations of 10 µg mlTo identify genes and signatures whose expression was significantly different between paired samples  or unpaired samples  we used two-class paired Significance Analysis of Microarrays (SAM) with a false discovery rate (FDR) <5%. All statistical tests were two sided, and the statistical significance level was set to <0.05.Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary


N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.The study of protein  N-linked glycosylation is one of the most common post-translational modifications of proteins.2+ from HT29 human colorectal cancer cell line N-glycans released by in-solution PNGaseF.Fig Z. Fragment ion spectra of precursor m/z 1276.57, [M + PROC]+ from HCT15 human colorectal cancer cell line N-glycans released by in-solution PNGaseF.Fig AA. Fragment ion spectra of precursor m/z 814.99, [M + PROC]+ from HCT116 human colorectal cancer cell line N-glycans released by in-solution PNGaseF.(DOCX)Click here for additional data file.


TCF3‐HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t‐ALL case, while TCF3‐PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t‐ALL cases. Using 4 t‐ALL and 16 t‐ALL cell lines, drug response profiling was analyzed. t‐ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t‐ALL cell lines. Sensitivities to three , four  and five  agents, widely used in induction therapy, were significantly poorer for t‐ALL cell lines than for t‐ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P‐glycoprotein (P‐gp) were higher in t‐ALL cell lines than in t‐ALL cell lines. Inhibitors for P‐gp sensitized P‐gp‐positive t‐ALL cell lines to VCR and DNR. Knockout of P‐gp by CRISPRCas9 overcame resistance to VCR and DNR in the P‐gp‐positive t‐ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P‐gp‐positive t‐ALL cell line. These findings indicate involvement of P‐gp in resistance to VCR and DNR in Pgp positive t‐ALL cell lines. In all four t‐ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t‐ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t‐ALL.t, responsible for TCF3‐HLF fusion, is a rare translocation in childhood B‐cell precursor acute lymphoblastic leukemia(BCP‐ALL). t, producing TCF3‐PBX1 fusion, is a common translocation in childhood BCP‐ALL. Prognosis of t‐ALL is extremely poor, while that of t‐ALL has recently improved dramatically in intensified chemotherapy. In this study,  Prior to analyses of the cell lines, we prospectively examined the levels of minimal residual disease (MRD) during induction therapy in a newly diagnosed t‐ALL case and t‐ALL cases. We confirmed high level of MRD in the t‐ALL case, indicating that ‐ALL shows resistance to chemotherapeutic agents in induction therapy. To verify this idea, we analyzed in vitro sensitivities to multiple chemotherapeutic agents of the t‐ALL and t‐ALL cell lines in induction therapy. We established that t‐ALL cell lines were significantly more resistant to VCR, daunorubicin (DNR), and glucocorticoids than t‐ALL cell lines. Furthermore, we confirmed comprehensive drug resistance to multiple agents in t‐ALL cell lines in induction therapy.22.1TCF3‐PBX1 was performed using sense (5′‐CCAGCCTCATGCACAACCA‐3) and antisense (5’‐ GGGCTCCTCGGATACTCAAAA‐3′) primers with probe (5′‐FAM‐CCCTCCCTGACCTGTCTCGGCC‐TAMRA‐3′), as previously described.2, 0.01 U Uracil DNA glycosylase per µL, 0.025 U of AmpliTaq Gold per microliter, and 1 × TaqMan PCR buffer . Amplification and detection were performed using an ABI 7900 sequence detection system . The data were analyzed by Sequence Detector version 1.63 software (Applied Biosystems). The final results were normalized by the amount of internal control GAPDH (Thermo Fisher Scientific). Real‐time PCR analysis for TCF3/HLF was performed using sense (5′‐GCCTCATGCACAACCACGCG‐3) and antisense (5′‐CCCGGATGGCGATCTGGTTC‐3′) primers with a SYBR Green PCR Master Mix (Applied Biosystems). As an internal control for TCF3/HLF quantification, ACTB was quantified using sense (5'‐ACCTTCTACAATGAGCTGCGT‐3') and antisense (5'‐GTACATGGCTGGGGTGTTGA‐3') primers.The patients were enrolled in Tokyo Children's Cancer Study Group (TCCSG) L04‐16 study.2.2MLL‐rearranged (MLL+) ALL cell lines , six Philadelphia chromosome (Ph)‐positive ALL cell lines , and eight other ALL cell lines  were used ‐ALL cell lines  and 16 t‐ALL cell lines  were used in this study.‐ALL celled Table .20 All c2.350s of DNR, VCR, prednisolone (Pred), dexamethasone (Dex), l‐asparaginase (l‐Asp), cyclophosphamide (CPM), and selumetinib, an alamarBlue assay was performed.5) were plated onto a 96‐well flat‐bottom plate in triplicate in the absence or presence of seven concentrations of each drug. The cells were cultured for 44 hours to determine the DNR, VCR, and CPM sensitivities and for 68 hours to determine Pred, Dex, l‐Asp, and selumetinib sensitivities, and, then, 20 µL of alamarBlue was added. After a 6‐hours additional incubation with alamarBlue, absorbance at 570 nm was monitored by a microplate spectrophotometer using 600 nm as a reference wavelength. Cell survival was calculated by expressing the ratio of the optical density of the treated wells to that of the untreated wells as a percentage. The concentration of agent required to reduce the viability of the treated cells to 50% of the untreated cells was calculated, and the median of three independent assays was determined as IC50. The median of the IC50s measured by three independent assays was determined.To determine IC2.4To detect apoptotic events, cells were cultured in the absence or presence of DNR or VCR in combination with or without verapamil, cyclosporine A (CyA), or nilotinib for 24 hours, and stained with a fluorescein isothiocyanate‐conjugated Annexin‐V  and actinomycin‐D . Cell surface expression of P‐gp was analyzed using a phycoerythrin‐conjugated anti‐P‐gp antibody. For the functional assay of P‐gp‐mediated efflux of calcein‐AM (CAM), HALO1 cells were incubated with 0.25 mmol/L of CAM for 10 minutes at 37°C in the absence or presence of velapamil, CyA, or nilotinib. The stained cells were analyzed by flow cytometry .2.5l‐Asp), four , and five  drugs were analyzed according to previous reports.50 values; seven cell lines), intermediately sensitive , or resistant  to each drug. A sensitive result was rated as 1, an intermediate result as 2, and a resistant result as 3, and the total score was calculated by adding these counts.The combined sensitivities of 4 t‐ALL and 16 t‐ALL cell lines to three , reverse transcription was performed using a random hexamer  by Superscript II reverse transcriptase (Invitrogen), and then incubation with RNase (Invitrogen). For quantitative real‐time PCR, triplicated samples containing cDNA with TaqMan Universal PCR Master Mix (Applied Biosystems) and Gene Expression Product listed in Table 2.74) were injected into the tail vein to establish xenografts. One day after injection of HALO1 cells, each of the five mice were treated with 0.5 mg/kg of DNR alone or 0.5 mg/kg of DNR in combination with 50 mg/kg of CyA for five consecutive days. DNR and CyA were further diluted with phosphate‐ buffered saline (PBS) and intraperitoneally injected into the mice. CyA was injected one hour before administration of DNR. The control group of five mice was administered PBS only.Six‐week‐old female NSG (NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ) mice were purchased from Jackson Laboratory . The experiment was performed in a specific pathogen‐free unit after approval of protocols for animal care and experiment by the Tokyo Medical and Dental University animal care and use committee (approved No. A2017113). HALO1 cells (1 x 102.8http://crispr.mit.edu), and the synthesized oligomers were cloned into CRISPR/Cas9 vectors . Three days after electroporation of the ABCB1‐targeting CRISPR/Cas9 vector into HALO1 cells using the Neon electroporation transfection system (Thermo Fisher Scientific), CD4‐positive cells were selected using CD4 microbeads  and expanded for further analyses.To knockout P‐glycoprotein  expression with the CRISPR/Cas9 system, we selected 5′‐TTTGGCTGCCATCATCCATGG‐3′, which showed the highest off‐target hit score in the CRISPR design tool . Libraries were prepared using the HaloPlex Target Enrichment System (Agilent Technologies), followed by paired‐end sequencing on a MiSeq instrument . Bioinformatic analysis was performed using the SureCall software (Agilent Technologies). Common germline polymorphisms reported in public databases were excluded and nonsense, frameshift, splice site, nonsynonymous variants were considered as mutations. Minimal allele frequency for mutation calling was set at 0.3.Target deep sequencing of RAS pathway genes including the 33.1TCF3‐HLF .We prospectively evaluated MRD levels in a t17;19)‐ALL case, treated with a high risk (HR) regimen in the TCCSG L04‐16 study,7;19‐ALL 3.2l‐Asp, and CPM) used in the induction therapy of the HR regimen of the TCCSG L04‐16 study. We determined the IC50 of each agent based on the dose‐response curve in an alamarBlue cell viability assay ‐ALL cell lines (median: 300 ng/mL) is significantly higher than that of t‐ALL cell lines (median: 15 ng/mL) (P = 0.006 in Mann‐Whitney test)   ‐ALL cell lines tended to be higher than that in t‐ALL cell lines ‐ALL cell lines in comparison with t‐ALL cell lines ‐ALL case in comparison with the t‐ALL cases. To verify resistance of t‐ALL to induction therapy in vitro, we analyzed the sensitivities of four t‐ALL cell lines, as well as 16 t‐ALL cell lines, to six agents  Figure B. IC50s ) Figure B and Pre) Figure C in t than those in the DNR‐treated t‐ALL cell lines (22%)  than those in the VCR‐treated t‐ALL cell lines (55%) ‐ALL and seven representative t‐ALL cell lines. When treated with 50 ng/mL of DNR, cell viabilities in two t‐ALL cell lines (UOCB1 and HALO1) and two t‐ALL cell lines (KOPN60 and 697) were 80%, 83%, 6%, and 4%, respectively Figure A. A simi) Figure C. Simila) Figure C.3.3l‐Asp), four  and five  agents ‐ALL cell lines to multiple agents used in induction therapy, we analyzed the combined sensitivities to three ‐ALL and t‐ALL cell lines were closely correlated with that of VCR  , MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), MRP6 (ABCC6), and BCRP (ABCG2). Among nine genes, MRP3, MRP5, and MRP6 were undetectable in BCP‐ALL cell lines. Gene expression levels of ABCB1 were significantly higher in t‐ALL cell lines than in other BCP‐ALL cell lines including the t‐ALL cell lines ‐ALL cell lines than in other BCP‐ALL cell lines, although they were almost equal between the t‐ALL cell lines and t‐ALL cell lines ‐ALL cell lines and other BCP‐ALL cell lines. Since ABCB1 gene expression levels were significantly higher in t‐ALL cell lines, we next analyzed the cell surface expression of P‐gp using flow cytometry. Among four t‐ALL cell lines, P‐gp was clearly detectable in HALO1 and UOCB1 and marginally detectable in Endo‐kun, but almost undetectable in YCUB2  in 32 BCP‐ALL cell lines. Cell surface expression level of P‐gp was relatively higher in t cell lines than in t‐ALL cell lines (P = 0.088 in Mann‐Whitney test)  Figure A. Since s Figure . No sign3.5To test the involvement of P‐gp in resistance to DNR and VCR in t‐ALL cell lines, we evaluated the functional drug‐efflux activity using calcein‐AM (CAM), an ABC transporter‐dependent dye.3.6P = 0.018 in Kaplan‐Meier analysis).We finally tried to confirm the involvement of P‐gp in DNR resistance of t‐ALL in vivo using NSG mice. After inoculation of HALO1 cells into NSG mice, we treated mice with DNR alone or DNR in combination with CyA for 5 days  in t‐ALL and t‐ALL cell lines using a next generation sequencer ‐ALL cell lines, respectively, and gene mutation in RAS pathway was detectable in all t‐ALL cell lines. In contrast, mutations in PTPN11, NRAS, KRAS, and NF1 genes were detectable in none, three, four, and one of 16 t‐ALL cell lines, respectively, and gene mutation in RAS pathway was detectable in seven out of 16 t‐ALL cell lines. Incidence of RAS pathway mutation tended to be higher in t‐ALL cell lines than in t‐ALL cell lines (P = 0.094 in chi‐square test).Association of gene mutation in the RAS pathway with poor therapeutic outcome in childhood ALL is controversial.er Table A. Mutati3.8KRAS mutation are sensitive to inhibitors of MAP kinases in vitro.KRAS mutation. We determined IC50 of selumetinib in t‐ALL and t‐ALL cell lines using an alamarBlue cell viability assay ‐ALL cell lines (two cell lines with KRAS mutation and two cell lines without it) (median: 100 μmol/L) was almost identical to that in 16 t‐ALL cell lines (four cell lines with KRAS mutation and 12 cell lines without it) (97 μmol/L). Of note, six cell lines with KRAS mutation [two t‐ALL cell lines and four t‐ALL cell lines] were relatively more sensitive to selumetinib than 14 cell lines without KRAS mutation [two t‐ALL cell lines and 12 t‐ALL cell lines] (P = 0.060 in Mann‐Whitney test).A recent report revealed that ALL samples with y Figure B. The IC3.9l‐Asp than those without it (P = 0.050 in Mann‐Whitney test). Then, we compared the total score of three, four, and five drug sensitivities of t‐ALL cell lines with RAS pathway mutation with those without it .We finally analyzed a possible association of RAS pathway mutation with drug resistance in t‐ALL and t‐ALL cell lines, since RAS pathway mutation was observed more frequently in t‐ALL cell lines. Association of RAS pathway mutation with sensitivity to each of the five drugs was not statistically significant in t‐ALL cell lines Figure , but t‐ALL during intensified induction therapy. Fischer et al have reported that MRD remains positive at the end of induction therapy in most t‐ALL cases.50s of Pred, VCR, and DNR in four t‐ALL cell lines were significantly higher than those in 16 t‐ALL cell lines. We also confirmed that the combined sensitivities to three , four , and five  agents in t‐ALL cell lines were significantly higher than in t‐ALL cell lines. These observations suggest that resistance to multiple agents, in particular to Pred, VCR, and DNR, may be associated with poor response to induction therapy of t‐ALL.In this study, case of t;19‐ALL d50s of DNR and VCR that are sensitive to ABC transporters are correlated with each other in t‐ALL and t‐ALL cell lines. Consistently, among eight genes of ABC transporters, the gene expression level of ABCB1 was significantly higher in t‐ALL cell lines than in t‐ALL cell lines. We also found that cell surface expression of P‐gp tended to be higher in t‐ALL cell lines than in t‐ALL cell lines, suggesting that P‐gp expression may be involved in resistance to VCR and DNR of P‐gp‐positive t‐ALL cell lines. P‐gp was active in P‐gp‐positive t‐ALL cell lines, as we found that inhibitors for P‐gp such as CyA and nilotinib intensified CAM staining and antileukemic activities of VCR and DNR. More precisely, we confirmed that knockout of P‐gp expression by CRISPR‐Cas9 overcomes resistance to VCR and DNR in a P‐gp‐positive t‐ALL cell line. We additionally confirmed that a combination of CyA with DNR significantly prolonged survival of NSG mice inoculated with a P‐gp‐positive t‐ALL cell line in comparison with DNR alone. Although in vivo combination activities of nilotinib and verapamil with DNR were not directly tested, these findings strongly suggest that overexpression of P‐gp is involved at least partly in resistance to VCR and DNR of P‐gp‐positive t‐ALL cell lines. Baudis et alABCB1 gene expression is detectable by RT‐PCR in Reh cells transfected with TCF3‐HLF under the influence of the zinc‐inducible promoter, suggesting a possibility that the ABCB1 gene is one of the downstream target genes of TCF3‐HLF.To verify the underlying mechanism(s) for poor response of t‐ALL to VCR and DNR, we focused on ABC transporters,NR3C1) gene (specific for GRα and GRγ isoforms), shows significant correlation with IC50 of Pred in 72 BCP‐ALL cell lines.38 However, an association of higher P‐gp expression with resistance to glucocorticoids is unlikely at least in HALO1 cells, since knockout of P‐gp expression by CRISPR‐Cas9 did not overcome Dex resistance.In addition to VCR and DNR, t‐ALL cell lines were significantly more resistant to Pred than t‐ALL cell lines. Sensitivity of ALL cells to glucocorticoids is highly associated with expression of the glucocorticoid receptor (GR). Our previous analyses demonstrated that the gene expression level of GR, analyzed by real‐time RT‐PCR of exons 8 and 9a of GR ‐ALL cell lines and 12 t‐ALL cell lines without the mutation. This higher sensitivity to selumetinib seems to be consistent with the above hypothesis that RAS pathway mutation may provide an advantage for in vitro cell growth and/or cell survival of the cell lines. Furthermore, among 16 t‐ALL cell lines, multidrug resistance was significantly more common in the cell lines with RAS pathway mutation than those without it. These observations suggest that frequent RAS pathway mutation may be involved at least partly in the aggressive clinical course of t‐ALL.A previous report revealed that genes in RAS pathway are frequently mutated in clinical samples of t17;19)‐ALL cases but not in t‐ALL cases.;19‐ALL cIn summary, our observations of a large panel of cell lines revealed that t‐ALL cell lines were significantly more resistant to multiple agents in induction therapy in comparison with t‐ALL cell lines. Although there are some limitations in using the cell lines in drug sensitivity studies, our findings seem to be consistent with the clinical notion that t‐ALL is resistant to intensified induction therapy in comparison with t‐ALL. Thus, these cell lines may be optional tools to study the mechanism(s) for drug resistance and to verify the activities of newly developed compounds in t‐ALL.The authors declare no competing financial interests.AW performed the research, analyzed the data, and wrote the paper; T. Inukai designed the research study, performed the research, analyzed the data, and wrote the paper as a principal investigator; KK, MA, MT, TF, HF, TN, KT, T. Ito, TT, DH, TS, SS, HO, KA, and K.G performed the research; JF, HG, ME, TL, MK, MM, JK T.Inaba, and HT contributed essential samples and cell lines; EI, AO, and KS supervised the project; and all authors contributed to the final draft. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Nepeta crispa essential oil was compared with propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines.Attempts to use the plant products are to be an appropriate option due to substantial concerns about human health and environmental problems of using synthetic pesticides. Therefore, the cytotoxicity of Spodoptera frugiperda (Lepidoptera: Noctuidae) and mouse fibroblast cells, respectively, were obtained from the National Cell Bank of Pasteur Institute of Iran. About a number of 2 × 103 cells were placed into the wells of 96-well plate experiments. Then, appropriate concentrations of essential oil of N. crispa plant and propoxur added to the wells. The cells were allowed to grow for 3-5 days and estimated the numbers of cells. The cells of control experiment wells contained only cells with dimethyl sulfoxide. All control and treatment experiments repeated at least four replicates.The cell lines of Sf9 and L929 which were derived from the ovary glands of fall armyworm, N. crispa essential oil against vertebrate (L929) cell line was gradually decreased in accordance with plant concentrations, while the cytotoxicity of N. crispa essential oil against invertebrate (Sf9) cell line was strongly increased in accordance with plant concentrations.Propoxur had negative effects on the viability of both invertebrate (Sf9) and vertebrate (L929) cell lines. The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased in accordance with propoxur concentrations. The cytotoxicity of N. crispa plant had negative effects on invertebrate cell viability with the differences that the products derived from plants possessing of biodegradable and environmentally friendly derivatives, hydrolyzing rapidly in nature, and nearly having no destructive effects on environment, humans, or the mammals.Plant essential oil not only had no negative effects but also had boosting effects on vertebrate cell viability. Essential oil of  Attempts to use the plant products as natural pesticides usually based on plant essential oils are considered as a good candidate for the management of arthropod vectors of diseases and agricultural pests due to possessing of readily biodegradable and environmentally friendly derivatives -5. ThereThere are substantial concerns about human health and environmental problems of synthetic pesticides. Pesticides have potential side effects despite their importance to public health. Exposures to various types of insecticides may be a risk factor for cancers. Several undesirable effects of pesticides are maybe toxicity to non-target organisms, residues of pesticides, depletion of ozone layer, environmental pollution, and direct toxicity to users ,9-12. InNepeta crispa Willd.  that is popular in Iranian traditional medicine, especially with Hamadan Province people. N. crispa have insecticidal activity with antimicrobial and antifungal properties. The N. crispa plants are autochthonous of Iran climate, especially Hamadan Province [One of the most natural aromatic plants in Iran is Province -22.N. crispa plant, but a simultaneous comparative study about the cytotoxicity of essential oil of N. crispa with synthetic pesticides such as propoxur against the cell lines of invertebrates and vertebrates would be a particular of importance. Therefore, in this study, we compared the cytotoxicity of N. crispa essential oil with propoxur against invertebrates and vertebrates cell lines.Many studies have been conducted on the insecticidal properties of The study was approved by the Ethics Committee of Hamadan University of Medical Sciences, Hamadan, Iran, with approval number: IR.UMSHA.REC.1396.320.Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Cell line of Sf9 is routinely cultured and maintained at 27°C in 5 ml of Grace’s insect cell culture medium in 25 cm2 culture flasks, enriched with 10% fetal bovine serum at Pasteur Institute of Iran. The doubling time of Sf9 cells was found to be 18-24 h under optimum conditions. Sf9 invertebrate cells were subcultured every 3 days. Cell line of L929 was derived from mouse fibroblast cells. It was maintained at 37°C in 3 ml of Dulbecco’s Modified Eagle Medium Media (Gibco®) in 25 cm2 culture flasks, enriched with 10% fetal bovine serum, and buffered with 4% sodium bicarbonate in an atmosphere of 5% carbon dioxide. The doubling time of L929 cells for cultures was approximately 24 h and the cell was subcultured every 6 day [Invertebrate (Sf9) and vertebrate (L929) cell lines were obtained from the National Cell Bank of Pasteur Institute of Iran. Cell line of Sf9 derived from the ovary glands of fall armyworm, ry 6 day .N. crispa  during their flowering stage were collected from Avicenna Medicinal Herbs Research Center, Hamadan Province of Iran in June 2017. The plant was confirmed by a voucher specimen (no. 72) in the Department of Pharmacognosy, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran. A total of 1000 g powder of shade-dried aerial parts of N. crispa were subjected to hydrodistillation using a Clevenger-type apparatus for 4 h. The essential oil was dehydrated over anhydrous sodium sulfate and transferred into amber-colored vials to store in a refrigerator at 4°C for further work.Technical grade of propoxur  was purchased from Guangdong Company, China (Mainland). The aerial parts (foliage) of N. crispa plant was dissolved with 1 ml of dimethyl sulfoxide (DMSO) due to hydrophobic properties and then diluted with sterilized distilled water to prepare the concentrations of 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, and 10−3 ng/μL containing 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng/μL, respectively. Propoxur technical grade (97%) was dissolved with ethanol 96% to prepare the concentrations of 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, and 10−3 µg/μL containing 0.000097, 0.00097, 0.0097, 0.097, 0.97, 9.7, 97, and 970 µg/μL, respectively.Essential oil (0.1 mg) of N. crispa plant and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines, about 2 × 103 cells/100 µl of culture medium were placed into the wells of a 96-well plate of treatment experiments and then appropriate concentrations of N. crispa essential oil and propoxur added to the wells. We allowed the cells to grow for 3-5 days and estimated the number of cells as described. Control cell wells contained only cells with 1µl/mL of DMSO. All treatment and control experiments repeated at least four replicates.To consider the cytotoxicity of essential oil of in vitro cytotoxicity which measures the active metabolism of the cells. In this coloring solution, dehydrogenase enzyme reduced the MTT and produced blue formazan.The base method for cell estimation is the Mossman method, which uses 3--2,5-difenyltetrazolium bromide . MTT is a quantitative coloring for living cells and cell proliferation, and it is a known method for The wells of 96-well plates containing invertebrate (Sf9) and vertebrate (L929) cell lines were incubated with 10 µ MTT for 3 h at 36°C and 27°C, respectively. After the blue formazan and cells settled out and the supernatant was removed, 100 µ of DMSO was added to any well of 96-well plate, shaked for 15 min and then the absorbance of the solution read at 492 nm using ELISA reader .N. crispa essential oil and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines were estimated by Microsoft Excel version 2013. The trend lines were drawn by clicking on graph line distribution and selecting “add trendline” option using procedure described in previous studies [N. crispa plant and propoxur between control and treatments, and cross treatments of N. crispa plant and propoxur. p<0.05 was considered statistically significant.The cytotoxicity trend lines of  studies -28.The eN. crispa essential oil and propoxur cytotoxicity against invertebrate (Sf9) and vertebrate (L929) cell lines, respectively. Figures-N. crispa essential oil and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines, respectively. N. crispa essential oil (ng/μL) and propoxur (μg/μL) against invertebrate (Sf9) and vertebrate (L929) cell lines. N. crispa plant, and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines.Tables-ebrate L99 cell liN. crispa plant against invertebrate (Sf9) cell line was strongly increased with an intensive increasing slope in accordance with essential oil concentrations of N. crispa plant from 10−10 to 10−3 µg/μL  cell line was gradually decreased with a moderately increasing slope in accordance with essential oil concentrations of N. crispa plant from 10−10 to 10−3 µg/μL  cell line with control (p<0.05)  cell line with control (p=0.068)  cell line with control  cell line with control (p>0.05)  . Wilcoxop=0.068) , even th control . Wilcoxo(p>0.05) , even the of Sf9 .−10 to 10−3 µg/μL  cell line with control (p>0.05) (−10 and 10−5 to 10−3 concentrations of propoxur against invertebrate (Sf9) cell line (−10 and 10−3 concentrations of propoxur against vertebrate (L929) cell line with control (p<0.05) (The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased with a relatively low decreasing slope in accordance with propoxur concentrations from 10 Figures-, b and 3(p>0.05) , even thell line . There w(p<0.05) .N. crispa plant against invertebrate (Sf9) cell line was strongly increased with an intensive increasing slope in accordance with essential oil concentrations of N. crispa plant, while the cytotoxicity of essential oil of N. crispa plant against vertebrate (L929) cell line was gradually decreased with a moderately increasing slope in accordance with essential oil concentrations of N. crispa plant. The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased with a relatively low decreasing slope in accordance with propoxur concentrations  and vertebrate (L929) cell lines, and invertebrate (Sf9) cell line, respectively (p>0.05), even though there were significant differences between cross treatments (10−10–10−4) of N. crispa plant and propoxur concentrations against vertebrate (L929) cell line (p<0.05) (Wilcoxon signed-rank test did not show significant differences between separatel(p<0.05) .−5-10−3 and 10−8-10−3 concentrations of propoxur with control group against the invertebrate (Sf9) and vertebrate (L929) cell lines, respectively, at p<0.05 or p<0.001 levels  and vertebrate L929) cell lines. These results confirm the reports of the previous studies that concluded insecticides decreased growth of the invertebrate and vertebrate cell lines  cell lin. Compare1 levels .N. crispa plant did not have negative effects on the viability of vertebrate (L929) cell line. Compared with the control group, the highest rate of cell viability was observed in the experiment treatments which were treated with essential oil of N. crispa plant. The viability of vertebrate (L929) cell line which was exposed to different essential oil concentrations of N. crispa plant was higher than control group and increased with increasing essential oil concentrations of N. crispa plant. The boosting effects of experiments of N. crispa plant on the viability of vertebrate (L929) cell line were also confirmed by Wilcoxon signed-rank test by observing significant differences between the treatments of 10−10 and 10−3 essential oil concentrations of N. crispa plant with propoxur treatments of 10−10-10−3 concentrations and control group against the vertebrate (L929) cell line at p<0.05 or p<0.001 levels. In addition to some previous benefits of the natural products deriving from natural plants [N. crispa plant on the viability of cell line of vertebrates may be considered as the new benefits of the products deriving from natural plants.Unlike propoxur, essential oil of l plants ,32, thisN. crispa plant had negative effects on the viability of invertebrate (Sf9) cell lines with the differences that the products deriving from natural plants possessing of readily biodegradable and environmentally friendly derivatives, hydrolyzing rapidly in nature, and nearly having no destructive effects on environment, humans, or the mammals [N. crispa plant (ng/μL) was 1000 folds lower than concentrations of propoxur (μg/μL). The viability of invertebrate (Sf9) cell lines which were exposed to essential oil concentrations of 10−9 and 10−7-10−3N. crispa plant was lower than control group and decreased with increasing essential oil concentrations of N. crispa plant.Like propoxur, essential oil of  mammals ,2. In adN. crispa plant on the viability of invertebrate (Sf9) cell lines were also confirmed by Wilcoxon signed-rank test by observing a significant difference between the treatments of 10−7 and 10−3 essential oil concentrations of N. crispa plant with control group against the invertebrate (Sf9) cell lines at p<0.001 level. In addition to that, the food chain and ecosystem contamination with pesticides, non-target organism extinction, insecticide resistance of insects, mutation, and pollution of water and soil have been critical problems using pesticides in the past decades. However, with the application of the products deriving from natural plants, there is no concern about their potential side effects such as toxicity to non-target organisms, residues of pesticides, depletion of ozone layer, environmental pollution, and direct toxicity to users. We will not encounter probably some psychotic disorders, neurological diseases and fatigue of muscles or cancers, and face up to severe insecticide effects on performance of vertebrate reproductive system [The negative effects of essential oil of e system ,9-12. Soe system -19.N. crispa plant did not have negative effects on the viability of vertebrate cell line. The viability of vertebrate cell line was increased with increasing essential oil concentrations of N. crispa plant. This boosting effect of N. crispa plant on the viability of vertebrate cell line may be considered as the new benefits of the products deriving from natural plants.Based on the results of the study, propoxur had negative effects on the viability of both invertebrate and vertebrate cell lines. Unlike propoxur, essential oil of AS and AZ planned the project. MB and HN conducted the experimental laboratory work and analyzed the data. SS assisted in analyzing of experimental data. AS and HN wrote the manuscript. AS supervised the project. All authors discussed the results and implications and commented on the manuscript at all stages. All authors read and approved the final manuscript.


Mesua beccariana was carried out. A new cyclodione, mesuadione (1), along with several known constituents which are beccamarin (2), 2,5-dihydroxy-1,3,4-trimethoxy anthraquinone (3), 4-methoxy-1,3,5-trihydroxyanthraquinone (4), betulinic acid (5) and stigmasterol (6) were obtained from this ongoing research. Structures of these compounds were elucidated by extensive spectroscopic methods, including 1D and 2D-NMR, GC-MS, IR and UV techniques. Preliminary tests of the in vitro cytotoxic activities of all the isolated metabolites against a panel of human cancer cell lines Raji (lymphoma), SNU-1 (gastric carcinoma), K562 (erythroleukemia cells), LS-174T , HeLa , SK-MEL-28 , NCI-H23 (lung adenocarcinoma), IMR-32 (neuroblastoma) and Hep-G2 (hepatocellular liver carcinoma) were carried out using an MTT assay. Mesuadione (1), beccamarin (2), betulinic acid (5) and stigmasterol (6) displayed strong inhibition of Raji cell proliferation, while the proliferation rate of SK-MEL-28 and HeLa were strongly inhibited by stigmasterol (6) and beccamarin (2), indicating these secondary metabolites could be anti-cancer lead compounds in drug discovery.An investigation on biologically active secondary metabolites from the stem bark of  Mesua, which is from the Clusiaceae family is a prolific producer of metabolites. Different parts of Mesua species are traditionally used as folk medicine for treatment of dyspepsia, fever, renal diseases and even as a poultice  (12), 136 (12), 123 (20), 109 (33), 96 (82), 82 (100), 67 (80), 56 (10). For 1H- and 13C-NMR spectra, see Beccamarin (2). Yellow solid. m.p. 139.0–139.6 °C. UV (EtOH) λmax nm (log ε): 209, 229, 281 and 348. IR νmax (cm−1): 3400, 2971, 1741, 1605 and 1466. MS m/z (rel. int.): 406, 392, 377, 350, 349, 293, 43. The 1H and 13C-NMR (CDCl3) spectral data are consistent with published data [2,5-Dihydroxy-1,3,4-trimethoxyanthraquinone (3). Orange solid. UV (MeOH) λmax nm (log ε): 218, 276 and 410. IR νmax (cm−1): 3400, 2920, 2840, 1660, 1630, 1540. MS m/z (rel. int.): 330, 315, 312, 297, 287, 272, 227, 58. The 1H and 13C-NMR (CDCl3) spectral data are consistent with published data [4-Methoxy-1,3,5-trihydroxyanthraquinone (4). Orange solid. UV (MeOH) λmax nm (log ε): 279, 320, 425, 470 and 485. IR νmax (cm−1): 3420, 2920, 2860, 1720, 1630, 1470. MS m/z (rel. int.): 286, 268, 257, 243, 212, 180. The 1H and 13C-NMR (CDCl3) spectral data are consistent with the published data [hed data .Betulinic acid (5). White solid. m.p. 290–291 °C. IR νmax (cm−1): 2939, 1687, 1453, 1375. MS m/z (rel. int.): 456, 248, 207, 189, 121, 107, 95, 81, 69, 55. The 1H and 13C-NMR (CDCl3) spectral data are consistent with the published data [Stigmasterol (6). White needles. m.p. 165–166 °C. IR νmax (cm−1): 3399, 2939, 1457, 1374. MS m/z (rel. int.): 412, 255, 159, 145, 95, 81, 69, 55. The 1H and 13C-NMR (CDCl3) spectral data are consistent with the published data [2 T-flask and maintained at 37 °C in 5% CO2 humidified incubator. All cell lines were sub-cultured depending on the confluence of cells and observed under an inverted microscope for cell viability and checking for any contamination. Once the cells reach the confluent state, cell numbers were evaluated using the trypan blue staining method in a hemocytometer. All cell lines were deposited in American Type Culture Collection (ATCC). Besides, all the cell lines except for the Raji and HeLa were classified under the biosafety level 1 by ATCC.Nine cancer cell lines: Raji, SNU-1, LS-174T, HeLa, SK-MEL-28, NCI-H23, IMR-32, Hep-G2 and K562 were obtained from Universiti Tunku Abdul Rahman (UTAR). The Raji, SNU-1, K562, HeLa, Hep G2 and NCI-H23 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) while the LS-174T, SK-MEL-28 and IMR-32 were maintained in MEM supplemented with 10% FBS. All the cell lines were cultured in 75 cmThe cytotoxicity study was performed by the MTT assay as described by Mosmann . SelecteAn aliquot of 100 µL of each sub-stock with different concentrations were added to each well together with 100 µL of selected cells to give concentrations of 100.00, 50.00, 25.00, 12.50, 6.25 and 3.13 µg/mL and made up to a final volume of 200 µL in each well. Cells with no extracts  and 200 µL of medium only (blank medium—negative control) were prepared in the same plate. Samples and controls were prepared in triplicate.2 incubator. After 72 h, 20 µL of MTT solution was added to all the wells and incubated for 3 h in a 5% CO2 incubator. The plate was then spun at 3,000 rpm for 10 min. Supernatant from each well (160 µL) was discarded and then DMSO (160 µL) was added to dissolve the purple formazan crystals.The plate was then incubated for 72 h at 37 °C in 5% COThe absorbance of each well was determined using a microplate reader at 550 nm. The average absorbance of each crude extract was calculated and the average value was used to determine the percentage of cell viability by using the following formula:where: A = absorbance of sample       B = absorbance of negative control       C = absorbance of positive control50 values were obtained from the plotted graph. Further dilutions were only performed on the compounds with IC50 values less than 3.13 µg/mL. Three independent experiments were conducted to assure the accuracy of the results. Kaempferol and quercetin were used as standard compounds throughout the cytotoxicity experiments.A graph of percentage of cell viability versus concentration was plotted for each extract and pure compound. The IC1), was isolated from the stem bark of Mesua beccariana along with several known constituents 2–6. Compounds 1, 2, 5, and 6 were strongly cytotoxic towards the Raji cell line. These phytochemical and cytotoxicity investigations which demonstrated the cytotoxic effects of these compounds suggest them to be potential chemopreventive agents in drug discovery. A new cyclodione, mesuadione (


Acaromyces ingoldii FS121, which was obtained from the China South Sea, yielded a new naphtha-pyrandione analogue, acaromycin A (1) and a new thiazole analogue, acaromyester A (2), as well as the known compound (+)-cryptosporin (3). Their structures, including absolute configurations, were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) spectra. Compounds 1–3 were evaluated for in vitro growth inhibitory activities against four tumor cell lines , wherein compounds 1 and 3 exhibited considerable growth inhibitory effects, with IC50 values less than 10 µM.Activity-guided isolation of the fermentation broth of the deep-sea derived fungus  Marine organisms live in a biologically competitive environment with unique environmental conditions, therefore marine-derived fungi have been proven to be a rich source of structurally unique and biologically active secondary metabolites ,7,8,9,10Acaromyces ingoldii FS121 showed potent cell growth inhibitory effects against SF-268, MCF-7, NCI-H460 and HepG-2 human tumor cell lines , five sp2 quaternary carbons δC 162.2 (C-7), 155.2 (C-10a), 132.1 (C-9a), 118.8 (C-4a), 114.1 (C-5a), three sp2 methines δC 137.2 (C-6), 124.7 (C-8), 119.3 (C-9), three sp3 methines δC 76.2 (C-2), 66.9 (C-3), 65.1 (C-4), and one methyl δC 16.7 (C-11). As six of nine degrees of unsaturation account for four double bonds (eight sp2 carbons), and two carbonyl groups, the remaining degrees of unsaturation requires that 1 be a tricyclic system. The HMBC correlations (H 11.68) to C-5a, C-7 and C-8. Furthermore, the 1H-1H COSY spectrum -cryptosporin (3), except for the OH at C-9 in 3 was locked at C-7 in 1. Therefore, the planar structure of 1 was confirmed.Acaromycin A , H-3 , H-4 ] with those of 3 . This indicated that these protons were cofacial and designated as β-oriented, whereas the Me-11 was α-oriented in 1. The absolute configuration of 1 was determined by the CD spectrum. The absolute configuration of (+)-cryptosporin (3) was established by synthesis and its CD spectra data was reported in the literature . This assumption was further confirmed by the HMBC correlations from H-2 to C-1, C-3 and C-8, H-5 to C-3 and C-7, and H-8 to C-6 and C-4. Besides, the 1H-NMR of 2 also exhibited a methyl signal at δH 2.24 , two methylene protons at δH 3.02  and 3.84 , and an olefinic proton due to the thiazole ring at δH 8.61 , suggesting the presence of a 4-methyl-5-thiazoleethanol substructure in 2, which has been reported in the literature + 299.0533 .Acaromyester A (2): light pink oil; UV (MeOH) λmax (log ε) 202 (4.24), 225 (4.08), 277 (3.44) nm; IR νmax 3309, 3178, 1732, 1716, 1516, 1236, 1145, 804, 783 cm−1; 1H- and 13C-NMR, see m/z [M + H]+ 278.0845 .1–3 were tested against four tumor cell lines, including MCF-7 (human breast adenocarcinoma cell line), NCI-H460  and SF-268 (human glioma cell line), HepG-2 (human hepatomacarcinoma cell line) by the SRB method pyran-5,10-quinone and a new heterodimeric compound combining a hydroxyphenylacetate and a 4-methyl-5-thiazoleethanol substructure, which were isolated from the fermentation broth of Acaromyces ingoldii FS121. It merits attention that compounds 1 and 3 contained a 3,4-dihydro-2H-naphtho-pyran-5,10-quinone core framework, which exhibited a wide range of biological activities [1–3 were evaluated, wherein compound 1 and 3 showed considerable activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines.In summary, we have described herein the isolation and structural elucidation of a new 3,4-dihydro-2