Hypericum stellatum, a species endemic to China, is used to treat hepatitis by several ethnic groups in Guizhou Province. This research was inspired by the traditional medicinal usage of H. stellatum, and aims to explore the phytochemistry and bioactivity of H. stellatum to explain why local people in Guizhou widely apply H. stellatum for liver protection. In this study, two new prenylated xanthones, hypxanthones A (8) and B (9), together with seven known compounds, were isolated from the aerial parts of the plant. Spectroscopic data as well as experimental and calculated ECD spectra were used to establish the structures of these compounds. Six xanthones isolated in this study, together with four xanthones previously isolated from H. stellatum, were evaluated for their growth-inhibitory activities against five human liver carcinoma cell lines to analyze the bioactivity and structure-activity relationship of xanthones from H. stellatum. Isojacareubin (6) showed significant cytotoxicity against five human liver carcinoma cell lines, with an IC50 value ranging from 1.41 to 11.83 μM, which was stronger than the positive control cisplatin (IC50 = 4.47–20.62 μM). Hypxanthone B (9) showed moderate cytotoxicity to three of the five cell lines. Finally, structure-activity analysis revealed that the prenyl and pyrano substituent groups of these xanthones contributed to their cytotoxicity. Hypericum have a long history of use as herbal medicines in China. There are 64 Hypericum species (33 endemic) distributed throughout China  acetate (1.7 mg) and β-sitosterol (3.5 mg). Fr. 8 was subjected to silica gel CC using  to afford 10 fractions (Fr. D1–Fr. D10). Fr. D7 was fractionated by Sephadex LH-20  into three fractions (Fr. D7a–Fr. D7c). Fr. D7b was submitted to recycling preparative HPLC (CH3OH) to afford hypxanthone A (1.4 mg), hypxanthone B (1.2 mg), and daucosterol (4.5 mg).The powdered, air-dried aerial parts of The method used for ECD calculation has been previously reported . The ROE1H- and 13C-NMR data: see m/z 343.0826 [M − H]− .Hypxanthone A. Yellow amorphous powder; UV (MeOH) λmax (log ε) 327 (4.32) nm; −3 M, MeOH) λmax (∆ε) 210 (+33.3), 250 (+18.4), 326 (+3.5) nm; 1H- and 13C-NMR data: see m/z 393.1341 [M − H]– .Hypxanthone B. Yellow amorphous powder; H. stellatum against five human liver carcinoma cell lines , as well as the immortalized non-cancerous human liver cell (LO2) by the MTS method, in vitro, with cisplatin  and Taxol  as positive controls.Considering its purported medicinal value for the treatment of liver disease, we tested the cytotoxic activities of ten xanthones isolated from H-tetrazolium  assay  acetate, β-sitosterol, and daucosterol) were isolated and identified from H. stellatum. Ten xanthones  isolated from H. stellatum were tested for their cytotoxicity against five human liver carcinoma cell lines. Isojacareubin showed stronger cytotoxic activity than the positive control cisplatin; however, it was weaker than Taxol. Analysis of the structure-activity relationship suggested that xanthone derivatives bearing substituent groups such as prenyl and pyrano groups can improve cytotoxicity towards the tested cancer cell lines. These results indicated that H. stellatum is rich in xanthones. The indigenous knowledge leading local people in Guizhou to apply H. stellatum for liver protection and to treat hepatitis needs further research to reveal in order to reveal the mechanisms.In summary, two new compounds, hypxanthone A (


To date, the most common prediction strategies are based upon machine learning or other quantitative structure-activity relationships (QSAR) based approaches. In the present research, we propose and discuss a straightforward strategy not based on any learning modelling but exclusively relying upon the chemical similarity of a query compound to reference compounds with annotated activity against cell lines. We also compare the performance of the proposed method to machine learning predictions on the same problem. A curated database of compounds-cell lines associations derived from ChemBL version 22 was created for algorithm construction and cross-validation. Validation was done using 10-fold cross-validation and testing the models on new data obtained from ChemBL version 25. In terms of accuracy, both methods perform similarly with values around 0.65 across 750 cell lines in 10-fold cross-validation experiments. By combining both methods it is possible to achieve 66% of correct classification rate in more than 26000 newly reported interactions comprising 11000 new compounds. A Web Service implementing the described approaches (both similarity and machine learning based models) is freely available at:  The usual prediction approaches addressing this problem can be divided into two major groups:The prediction of a cell-line sensitivity to chemical compounds is a very important factor to optimize Approaches based on quantitative structure-activity relationships (Q)SAR modeling. These approaches aim at establishing a model relating chemical structures with their effect on different cell lines –6.Approaches based on gene expression such as the well-known CMAP and L1000CDS2 methodologies –9. OtherThe goal of the methods using gene expression is to compare the genetic expression profile triggered by a query compound with a known reference expression pattern generated with a particular cell line-compound pair. Therefore, these methods require the gene expression profile triggered by the compounds under investigation, which is frequently unknown.On the other hand, QSAR strategies had been previously applied for drugs-cell lines sensitivity prediction –6 with sThe core idea behind any QSAR model is the similarity principle. This principle postulates that compounds sharing a high structural similarity should also have similar activities/properties. Actually, the work of Zhang et al  clearly In the present work, we went through a direct similarity-based strategy being the first time that this approach is evaluated for predictions of compounds-cell lines interactions. Under this approach, no learning based algorithm is used to model the structure-activity space and only the consideration of molecular similarity is required. It is a simple and fast approach that was first applied in the area of chemicals-proteins interactions prediction and later coined as “target fishing” –17. In tWe first evaluated the influence of the radius of the MCFPs and similarity cutoff on the performance of our “cell fishing” or similarity-based approach. The results of these analyses are summarized in In The similarity-based strategy compares a query compound with known sensitive interaction for a particular cell lines. Therefore, the goal is to detect sensitive response. Increasing the similarity cutoff reduces the chances of finding similar molecules but if detected, the confidence will be higher Figs  and 2. AAs described in the methodology section, for SVM we choose cell lines with more than 20 compounds with reported sensitive and resistant interactions (a minimum of 40 compound per cell line). However, for the similarity-based approach we only need 10 sensitive compounds. This difference reduces the cells space from 960 cell lines and 109349 compounds with 140114 sensitive and 147690 resistant LCLAs to 758 cell lines and 107195 compounds with 134565 sensitive and 145064 resistant LCLAs.It can be noticed  that cloIn general, some sensitive/resistant association are better predicted with the similarity approach and vice versa. Moreover, several cell lines are equally modelled with both methods . By keepThe objective of both considered strategies is to identify the maximal amount of interactions between a query compound and all cell lines under consideration. Therefore, the goal in this experiment is to evaluate the capacity to identify all possible sensitive/resistant interactions for new molecules. In this sense, the metrics were calculated across the interactions space. This experiment is not comparable with cross-validation results because on the later the metrics were evaluated across cell lines. That is, in the 10-fold cross-validation the true positives ratio indicates the average detection across cell lines, instead of the true positive detection ratio in all known sensitive/resistant interactions for a given set of compounds as in .Three strategies were evaluated in external validation :The algorithm based on SVM over the 758 cell lines.The similarity-based algorithm over the 758 cell lines using the similarity cutoff of 0.20, 0.25 and 0.3.The combined SVM and similarity-based algorithms over the 758 cell lines using the similarity cutoff of 0.25.The similarity-based approach apparently lead to poor prediction compared to SVM. However, it is important to remember that the similarity-based strategy is based on comparing only to similar sensitive compounds. Therefore, it is possible that some of the new compounds cannot be compared to any of the existing ones in the database.For a cutoff of 0.20, the similarity approach covers almost 92% of the compounds, which means that for 10279 new compounds at least one cell line was predicted based on similarity. In this space, the true-positive and true negative ratios are comparable to those obtained by SVM. Actually, the inclusion of SVM models for the remaining 8% of the compounds do not improve performance , model 5The model 6  is the rIn the The ChemBL database is continuously growing regarding the number of assays as well as the number of new compounds. The addition of new compounds and biological assays, if SVM models are used for LCLAs predictions, will require the reevaluation of the entire model so scalability will become an important issue. In a similarity-based strategy, this problem can be simply addressed. In both cases, the compounds additions will lead to improvements in predictability because they will necessarily increase the molecular as well as the cell lines diversity.The machine learning strategy (as used in this work) provides several advantages including the learning-based extrapolation capabilities, which is noticed in the external data set evaluation. However, compared to the similarity approach, the latter is simplest and easily scalable. Even when both strategies are relatively close in terms of cross-validation, the evaluation of the external data indicates that new chemical scaffolds will be hard to predict using the similarity based strategy (coverage reduction). This is why better results were obtained combining both approaches. Our results also suggest that comparison, whenever possible, between advanced modelling techniques and the simpler strategies (in this case the direct similarity comparison) need to be considered.The prediction of a particular property using the chemical similarity principle is sensible to the description space used for modelling. In this research, we rely upon a comparison based on Tanimoto similarity measured over Morgan fingerprints. If for a particular cell-line only a small and conserved group of molecules is available, then the similarity will be small for any other query with a new molecular scaffold. Hence, better models (using machine learning and/or similarity-based) could be obtained including other descriptions of the molecular space combined with feature selection algorithms.cell_dictionary table distributed with ChemBL v22 in MySQL format was used to extract all cell identifiers (cell_id). Only those LCLA linked to assays reporting median inhibitory concentration (IC50), the median growth inhibitory concentration (GI50) and/or the median cytotoxic concentration (CC50) were considered for further processing. The canonical smiles of each compound was independently analyzed. In cases of salts or disconnected structures, only the largest fragment was used as the input structure. Next. LCLAs established at a ligand concentration lower than or equal to 10−5 M  were labeled as “sensitive”, and the rest were labeled as “resistant”. When more than one assay is reported for the same LCLA, it is removed if there is no total agreement between assays . Finally, any cell line sensitive to less than 10 compounds was removed from the final database. The combination of several assay types (ex. IC50. GI50 and CC50) into a single classification rule has been recently used by other authors in compounds-cell-lines sensitivity prediction [The ChemBL database version 22  was filtediction ,15.Accordingly, the final database included 960 cell lines comprising 140114 sensitive and 147690 resistant LCLAs. Details on the cell lines included in the final database can be found in the In this work. the target fishing approach proposed in Liu et al and recently applied by Peng et all ,23 was an closest sensitive compounds reported in the database for that cell line is higher than a certain similarity cutoff.To measure structural similarity, chemical structures were encoded using the Circular Morgan Fingerprints (CMFP), which are equivalent to the Extended Chemical Fingerprints (ECFP). implemented in RDKit . The Tanr = 2 (ECFP4). r = 4 (ECFP8). and r = 8 (ECFP16). The effect on predictability of different similarity cutoffs were explored. However. we should keep in mind the standard of chemical similarity established for most cheminformatics applications: a pair of molecules is considered as structurally similar if they share an ECFP4 Tc equals or higher than 0.55 [In this work, we tested n = 3 based on the average molecular similarity of the top 3 closest ligands. The efficiency of several similarity cutoffs for the CMFP defined by different radii in detecting sensitive associations was also explored. The evaluated similarity cutoffs ranged from 0.2 to 0.6 with a step size of 0.05. Three different radii of the CMFP were tested: han 0.55 .Machine learning (ML) classification models were evaluated with 758 cell lines out of the initial pool of 960 cell lines employed for target fishing. A total of 202 cell lines were excluded from these experiments because of having less than 20 compounds in any of the sensitive or resistant groups. The decision of a minimum of 20 compounds was based on Tropsha et al .To train binary classification models, the two groups need to be balanced in the number of compounds that each one contains. A dataset was considered unbalanced if the majority group contained more than 1.2 times the number of compounds in the minority one. From the 758 cell lines, 213 were balanced whereas the remaining 545 ones underwent a data balancing procedure before modeling.The data balancing protocol aimed at reducing the majority group to a similar number of compounds present in the minority class. The employed protocol relied in a hierarchical clustering procedure to retain the data diversity present in the initial majority group. For this, the chemical structures of the compounds in the majority group were coded using Morgan fingerprints of radius = 8 and 1024 bits. Then, the distance matrix between all pairs of compound belonging to the class to be reduced was computed employing the Tanimoto distance. The computed distance matrix was used as input to a tree-based clustering using the linkage function with the Ward’s minimum variance method. Clusters were defined using a cutoff height of 4.5.If the number of clusters (Nc) was lower than the number of compounds in the minority group (Nm), then a number of compounds was selected from each cluster, proportional to the cluster size, until completing Nm samples. This was the case for 112 cell lines. On the other side, if Nc > Nm, then the majority group was re-clustered to obtain Nm clusters and the centroid of each one was selected for the reduced dataset. This procedure was applied to 443 cell lines.The reduced datasets containing compounds coded with the Morgan fingerprints as described above were used to train Support Vector Machine (SVM) classification models with Radial Basis Function (RBF) kernels.jAC and NjRC are the number of compounds reported in the database as establishing a sensitive and resistant association respectively with cell line j.Several performance metrics, previously reported in Peng et all  for targThe described performance metrics were computed for two different validation strategies: 1) the 10-fold cross-validation and 2) new compound with known sensitive/resistant annotations in the same cell lines space.The SVM modeling was conducted using a 10-fold cross-validation setup. On each cross-validation cycle, 90% of the data was used to train a SVM model and the remaining 10% of it was employed as test set. Before training, the SVM C and γ parameters of the kernel were optimized by means of a grid search algorithm.In order to get a comparative space between machine learning strategy and the similarity-based approach, the same compounds used in each 10-fold iterations (therefore the same compound in the partition) were evaluated with both prediction methods. All metrics (if not reported otherwise) calculated for machine learning model and the similarity-based method were computed with the initial database of 758 cell lines and 107195 compounds with 134565 sensitive and 145064 resistant LCLAs.For the second type validation we carried on the same data treatment and filtering process already described for ChemBL v22 to ChemBL v25. With these new data we extracted the entire list of new compounds emerging from v22 to v25 and all their cell lines association (from the 758 cell lines): 11266 new compounds. 20898 sensitive pairs and 5237 resistant pairs .http://bioquimio.udla.edu.ec/cellfishing.Two strategies are available as a web-service in; The CellFishing (similarity-based strategy). All 960 cell lines are included. In this section different Morgan’s Radius can be used.The SVM models obtained in 758 cell lines.S1 Table(XLSX)Click here for additional data file.S2 Table(ZIP)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Alkaloids are used in traditional medicine for the treatment of many diseases. These compounds are synthesized in plants as secondary metabolites and have multiple effects on cellular metabolism. Among plant derivatives with biological properties, the isoquinoline quaternary alkaloid berberine possesses a broad range of therapeutic uses against several diseases. In recent years, berberine has been reported to inhibit cell proliferation and to be cytotoxic towards cancer cells. Based on this evidence, many derivatives have been synthesized to improve berberine efficiency and selectivity; the results so far obtained on human cancer cell lines support the idea that they could be promising agents for cancer treatment. The main properties of berberine and derivatives will be illustrated. Natural compounds have been used for centuries because of their availability; those present in plants are employed in the so-called Traditional Medicine, which translates theories, beliefs and experiences into knowledge, skills and practices applied to prevent, diagnose and treat physical and mental disorders . Being rPlants have wide biological and medicinal properties, and are characterized by high safety, availability, accessibility and low cost, thus representing an invaluable source of chemicals with potential therapeutic effects ,4. Secon20H19NO5, Hydrastis canadensis, Berberis aristata, Coptis chinensis, Coptis japonica, Phellondendron amurense and Phellondendron chinenseSchneid [in vitro studies using cancer cell lines have shown that BBR inhibits cancer cell proliferation and migration, and induces apoptosis in a variety of cancer cell lines [Among the several plant secondary metabolites, alkaloids possess a variety of pharmacological properties. Berberine .BBR interacts directly with nucleic acids and with several proteins, including telomerase, DNA topoisomerase I, p53, NF-kB, MMPs and estrogen receptors. In general, BBR treatment promotes cell cycle arrest and death in human cancer cell lines, coupled to an increased expression of apoptotic factors ,15,16. TSeveral studies have shown that BBR interacts directly with DNA, inducing double-strand breaks ,17, and 0/G1 phase was recorded in breast cancer MDAMB-231 and MCF-7 [0/G1 arrest and suppressing cell proliferation [1 phase, paralleled by decreased expression levels of cyclin B1, was observed in lung cancer H1299 and A549 cell lines [2 phase [1 (accompanied by p53-dependent upregulation of p21) and a p53-independent arrest in G2/M phase [wt were found to be very sensitive to BBR, whereas cell lines lacking functional p53 do not respond to BBR treatment [wt [Cell cycle arrest has been reported in human cancer cell lines as an effect of the interaction of BBR with DNA , as suppnd MCF-7 , thyroidnd MCF-7  and ovarnd MCF-7  cell linll lines  as well /M phase . In genereatment ,16. In tment [wt .2/M phase in colorectal, breast cancer and hepatocellular carcinoma cells followed by apoptosis activation through the loss of mitochondrial membrane potential, release of cytochrome c, inhibition of anti-apoptotic proteins , activation of pro-apoptotic proteins  and cleavage of PARP-1 [It was also observed that BBR induced cell cycle arrest at the Gf PARP-1 ,35,36,371/S boundary [2+ from endoplasmic reticulum (ER). An event which causes cell cycle arrest and cell death, driven by altered functions of mitochondria followed by cytochrome c release and caspase-3 activation leading to apoptosis [This protein  or DNA damage-inducible transcript 3 (DDIT3), is involved in growth arrest and DNA damage, ubiquitously expressed at very low levels  and overboundary ,39. It hpoptosis ,39,40,412), which are implicated in inflammatory responses and also in carcinogenesis and metastasis [2 [in vivo assays, BBR was found to lower COX-2 expression in the colon of rats treated with azoxymethane (AOM), inhibiting in turn the neoplastic transformation [Two COX isoforms with distinct physiological functions have been described; COX-1 is constitutively expressed and has an important role in cell homeostasis, while COX-2 is an inducible enzyme activated by extracellular stimuli. Overexpression of COX-2 stimulates the production of prostaglandins, including prostaglandin E2  regulates a wide range of cellular signaling pathways for both cell survival and death. In neuroblastoma cells it was observed that BBR binds to DNA in the DAXX core promoter region and suppressed its transcriptional activity. The down-regulation of DAXX expression resulted in the degradation of MDM2 (murine double minute 2) by ubiquitination, followed by the activation of p53 and then apoptosis ,16.It has been demonstrated that DAXX interacts with MDM2 and HAUSP (herpes virus-associated ubiquitin-specific protease) to form a tertiary complex. This complex reduces self-ubiquitination of MDM2, to maintain a low level of p53 under non-stress conditions. In different cancer cell types, BBR induced a dissociation of the MDM2-DAXX-HAUSP complex, resulting in enhanced MDM2 ubiquitination that increases p53 protein activity, leading to apoptosis .The AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine kinase that serves as a metabolic sensor for the maintenance of cellular energy homeostasis and is capable to inhibit AR (androgen receptor). AMPK becomes activated in cell starvation, hypoxia, ischemia and heat shock. In prostate cancer, AR signaling is crucial for development and progression by regulating cell proliferation, differentiation and apoptosis. After treatment with BBR, prostate cancer cells decrease their proliferation by a direct activation of AMPK, contributing to the degradation of AR and leading to apoptosis ,58.2H4PteGlu), generating dTMP and dihydrofolate [Thymidylate synthase (TS) catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate  and methotrexate, showed an increase in TS and in the intracellular pools of 5,10-methylene-THF and THF . These cThe search for new drugs that induce apoptosis in tumors refractory to the conventional therapy is crucial to develop efficient anticancer therapies. Several mechanisms by which BBR inhibits the proliferation of different cancer cell lines have been reported. Among them, the killing of cancer cells by the activation of apoptosis is the best characterized.In this context, several groups have reported the pro-apoptotic effect of BBR mediated by the impact on mitochondria. In fact, BBR was proved to alter the mitochondrial membrane potential (MMP), inhibit mitochondrial respiration leading to mitochondrial dysfunction and regulate the expression of Bcl-2 family members, as Mcl-1 ,47. AlteBBR pro-apoptotic effects could be mediated through the modulation of the HER2/PI3K/Akt ,72 and/oL after BBR treatment was observed not only in human prostate epithelial (PWR-1E) or carcinoma cells , but also in promyelocytic leukemia, gastric carcinoma and lung cancer cells, inducing cell death  together with decrease of Bcl-2/Bcl-xll death .Caspase-dependent apoptosis was reported in colon carcinoma cells treated with 13-arylalkyl BBR derivatives . BBR hasin vivo when tumor cells were injected into nude mice [BBR has been used in combination with drugs or radiation. It was found as an adjuvant therapeutic agent in combination with taxol, a frequently used clinical chemotherapeutic drug, in HER2-overexpressing breast cancer cells (SKBR-3) . Furtherude mice .in vitro could be related to the inhibition of FAK, IKK, NF-κB, u-PA, MMP-2, and MMP-9 [in vivo and in vitro analyses in B16F-10 cells, where BBR affected MMP expression through a negative regulation of ERK [Of note, BBR could interfere with tumor progression and invasion, possibly through the inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA), GTPase , PE2 recnd MMP-9 ,88,101. nd MMP-9 . The pron of ERK . The actn of ERK . The samn of ERK .Metastatic lung cancer A549 cells also appeared to be blocked in tumor progression and invasion by BBR, due to the down-regulation of several transcription factors such as c-fos, c-jun and NF-κB . The samAutophagy contrasts cellular stress conditions including nutrient deprivation and produces recycled energy originated from macromolecule degradation, thus regulating cellular homeostasis. The function of autophagy in cancer cells is to sense the presence of damaged DNA and organelles caused by chemotherapy and to degrade them, according to its own function; in this way, continuous energy is produced, which ensures cancer cell survival ,109,110.The analysis of the potential ability of BBR to induce autophagy revealed that in non-small-cell and Lewis lung cancer cell lines as well as mice with Lewis lung carcinoma xenografts, in fact autophagic hallmarks  were detected ,114. AccSeveral derivatives of BBR have been evaluated in the HCT116 colon carcinoma cell line and found to be capable of inducing vesicle formation at cytoplasmic level, where LC3I was converted into its lipidated form LC3II .Berberine offers ready functionalization and decoration at various positions on its skeleton, and can be readily converted into derivative compounds by known synthetic methodologies ,118,119.In particular, arylalkyl derivatives of berberine have been investigated ,119. UnpThe above discussed evidences suggest that multiple effects of BBR are mediated by the impact on different pathways, leading essentially to cell cycle arrest, apoptosis and controlled inflammation . The pro. The mechanism of action of miR-21-3p was postulated to be correlated to the expression of methionine adenosyltransferase (MAT) genes (MAT2A and MAT2B) [i.e., human multiple myeloma [Among the multiple studies aiming at defining the mechanism of action of BBR, it is noteworthy to mention the evidence that BBR could regulate microRNAs (miRNAs), short non-coding RNA molecules (21–23 nucleotides) generated in the nucleus and involved in a variety of biological processes, including development, cell proliferation and death. Deregulated expression of miRNAs was observed in many human cancer types, where they can act either as tumor suppressor or oncogenes . Recent d MAT2B) . In diff myeloma  and cisp myeloma , a dose


Pericharax heteroraphis collected from the South China Sea yielded one new imidazole alkaloid named naamidine J (1) along with four known ones (2–5). Their structures were established by extensive spectroscopic methods and comparison of their data with those of the related known compounds. All the isolates possessed a central 2-aminoimidazole ring, substituted by one or two functionalized benzyl groups in some combination of the C4 and C5 positions. The cytotoxicities against selected HL-60, HeLa, A549 and K562 tumor cell lines and anti-H1N1 (Influenza a virus (IAV)) activity for the isolates were evaluated. Compounds 1 and 2 exhibited cytotoxicities against the K562 cell line with IC50 values of 11.3 and 9.4 μM, respectively. Compound 5 exhibited weak anti-H1N1  activity with an inhibition ratio of 33%.Marine sponges continue to serve as a rich source of alkaloids possessing interesting biological activities and often exhibiting unique structural frameworks. In the current study, chemical investigation on the marine sponge  Pericharax heteroraphis (genus Pericharax family Leucettidae) drew our attention, as the UV characteristics of MeOH extracts showed the existence of 2-aminoimidazole alkaloids; in addition, its extracts were reported to have broad activity on cancer cell lines  ( (1) was ′, H-6′′ . Therefo2) amino}-1-methyl-1H-imidazole-2,5-dione. Yellow amorphous solid; melting point (m.p.): 102–104 °C; UV (MeOH): λmax (log ε) 225 (4.27), 275 (3.77), 392 (3.97) nm; IR (KBr): 3427, 2960, 2873, 1731, 1710, 1377, 1250, 1068, 1025 cm−1; 1H and 13C-NMR: see m/z 478.2091 [M + H]+ ; 500.1906 [M + Na]+ .Compound In vitro cytotoxicities were determined by MTT colorimetric assay against K562 (human leukemia cells) and HL-60 (human myeloid leukemia cells), SRB (Sulforhodamine B) assay against HeLa  and A549 (human lung adenocarcinoma cells). All the cell lines were purchased from Shanghai Institute of Cell Biology . Cytotoxic data  was used as a positive control, and IC50 values > 50 μM were considered to be inactive in cytotoxic assays.xic data  for comp2 and 95% air at 37 °C. Those cell suspensions (200 μL) at a density of 5 × 104 cell mL−1 were plated in 96-well microtiter plates and incubated for 24 h at the above conditions. The test compound solution (2 μL in DMSO) at different concentrations in triplicate was added to each well and was further incubated for 72 h under the same conditions. 20 μL of the MTT solution (5 mg/mL in IPMI-1640 medium) was then added to each well and incubated for 4 h. The old medium containing MTT (150 μL) was then gently replaced by DMSO and vibrated to dissolve any formazan [1-(4-Iodophenyl)-5-(4-nitrophenyl)-3-phenylformazan] crystals formed. The optical density of the solution was measured on a Spectra Max Plus plate reader at 570 nm. The IC50 value of each compound was calculated by Reed and Muench’s method.In the MTT assay , the cel5 cell mL−1. Then 2 μL of the test solutions (in MeOH) was added to each well, and the culture was further incubated for 24 h. The cells were fixed with 12% trichloroacetic acid, and the cell layer was stained with 0.4% SRB. The absorbance of the SRB solution was measured at 515 nm. Dose-response curves were generated, and the IC50 values (the concentration of compound required to inhibit cell proliferation by 50%) were calculated from the linear portion of log dose-response curves.In the SRB assay , 200 μL 50 was calculated as the compound concentration required inhibiting CPE production at 48 h post-infection by 50%. Ribavirin  was used as positive control, and compounds with an inhibition rate of >70%, >50%, and <30% at 50 μg/mL were respectively regarded as having strong, moderate, and weak activities.The antiviral activity against H1N1 was evaluated by the cytopathic effects assays (CPE) . Conflue1, 2, 3 and 5 from the genus Pericharax, and firstly reported anti-H1N1 virus activities of this type of alkaloid. Compounds 1 and 2 showed cytotoxic activity against the K562 cell line, suggesting that they might have the potential to be developed as antitumor agents. It was reported that the extract of Pericharax heteroraphis showed significant anti-AChE-inhibitory activity, and continuing investigation of the anti-AChE-inhibitory activity of the isolates is worthwhile.The present work offered one new member of 2-aminoimidazole alkaloids and the first isolation of compounds


Ovarian clear cell carcinoma is a distinct histologic subtype with grave survival. The underlying molecular mechanism is not fully elucidated. However, we don’t have many cell lines, which are useful experimental tools for research. We describe the establishment and characterization of a new ovarian clear cell carcinoma cell line from a Chinese patient.FDOV1 has been subcultured for more than 80 generations. Monolayer cultured cells are polygonal in shape, showing a transparent cytoplasm full of vacuoles. The number of chromosomes ranges from 45 to 90. FDOV1 cells produces CA-125, but not CA-199. The cells could be transplanted and produced tumors mimicking the donor tumor morphologically and immunohistochemically. Whole exome sequence showed both FDOV1 and tissue block harbored PIK3CA H1047R mutation and ARID1A frameshift mutations . More interestingly, we observed SPOP mutation (p.D82H) and ZNF217 (chromosome 20q13) amplification in FDOV1, which are quite novel.Only a few patient-derived ovarian clear cell carcinoma cell lines have been reported in the literature. FDOV1 is the very first one, to the best of our knowledge, from a Mainland Chinese patient. It showed infinite multiplication until now and tumorigenicity in vivo. FDOV1 has co-existing PIK3CA and ARID1A mutations. It also harbored SPOP mutation and ZNF217 amplification, which would probably be a good model for exploring the molecular mechanism of ovarian clear cell carcinoma.The online version of this article (10.1186/s13048-018-0429-5) contains supplementary material, which is available to authorized users. Epithelial ovarian carcinoma is the most lethal gynecologic malignancy. Ovarian clear cell carcinoma (OCCC) is the second most common histologic subtype, accounting for 5–25% of all ovarian cancer depending on geographic location , 2. It i-Rich Interactive Domain-containing protein 1A (ARID1A) and Phosphoinositide-3-kinase Catalytic Alpha (PIK3CA), representing around 50% [The two most widely reported gene mutations in OCCC are ATound 50%  and 40% ound 50% , 8 of alound 50% . What’s ound 50% , 11. Theound 50% , which shttp://www.atcc.org) and COSMIC database (https://cancer.sanger.ac.uk/cell_lines), respectively. The majority of the established cell lines were derived from Japanese patients. Besides, only one OCCC cell line TOV-21G was found with PIK3CA and ARID1A mutation. We describe here the establishment and characterization of a novel cell line (FDOV1) from a Chinese patient that harbors coexistent ARID1A-PIK3CA mutations.Cancer cell lines are affordable models for genetic and molecular profiles, reflecting the characteristics of the origin tumor. According to documentations till now, 16 OCCC cell lines were established and their characteristics have been reported –25, addeUp to now, more than 80 serial passages have been carried out successively. FDOV1 cells grew in the form of an adherent monolayer without contact inhibition ; 2n = 47 ; 4n = 86–90 . Chromosomal aberrations including complex translocations and deletions were noted, which were consistent with that of malignant tumors.A total of 45 metaphase cells from FDOV1 were examined for chromosome analysis. Chromosome numbers ranged from 45 to 90 , insertion and deletion (indel) and copy number variation (CNV). From the SNV related to cancer, it is shown that the tissue block and FDOV1 cell line shared some same somatic variations although diverse allele frequency Fig. . InteresOvarian clear cell carcinoma, as a subtype with distinct clinical behavior, is notorious for its poor survival and resistance to conventional platinum-based chemotherapy. An international phase III clinical trial was conducted to investigate the addition of irinotecan as adjuvant chemotherapy for patients with advanced-stage . UnfortuAccording to documentations, there were 16 ovarian clear cell carcinoma cell lines reported so far, in addition, TOV-21G, ES-2, which are commercially available from American Tissue Culture Center, and FDOV1. Their characteristics were compared in Table Notably, SPOP, a relatively new gene regulating DNA damage repair, is the most frequently mutated gene in prostate carcinoma , 33. MutChromosome 20q13 ZNF217 (Zinc Finger Protein 217) locus amplification, which has found in FDOV1, is one of common molecuclar genetic aberrations in OCCC . The fre. FDOV1 showed fast and unlimited multiplication until now and tumorigenicity in vivo. In addition, WES showed that it harbors concurrent ARID1A–PIK3CA mutations and SPOP mutation, also with ZNF217 amplification, which will be an ideal tool for mechanism research of ovarian clear cell carcinoma.An ovarian clear cell carcinoma cell line named FDOV1 was established from a Mainland Chinese patientA 67-year-old woman was admitted into Fudan University Shanghai Cancer Center with the chief complaint of palpable abdominal mass in May 2016. Ultrasound revealed a large pelvic mass (14 × 10 cm) with mixed components and a hyperechoic mural nodule (4 × 3 cm). Serum tumor markers including carbohydrate antigen (CA) 125 and CA199 were not elevated. Intraoperative findings were only significant for a cystic-and-solid mass (15 cm in diameter) in the left adnexa. Frozen pathology was highly suspicious of ovarian clear cell carcinoma. The patient underwent comprehensive staging surgery and the final stage was FIGO  Ia. She gave written informed consent before surgery according to institutional guideline (FUSCC 050432–4-1212B). Six cycles of TC regimen (paclitaxel + carboplatin) chemotherapy were administered. She was followed up every 3 months after treatment. The patient was admitted into outside hospital with chief complain of intestinal obstruction in May 2017. She developed multiple sites of venous thromboembolism and cerebral infarction. Unfortunately, she died 1 month later with no chance of anti-cancer treatment.3) from primary ovarian tumor was obtained during comprehensive staging surgery, and suspended in phosphate buffer solution (PBS) to get rid of necrotic and connective tissue. The material was then finely minced into 1-mm3 tissue blocks and placed in 10-cm cell culture dishes  containing Medium 199 medium  with 10% fetal bovine serum , 100 IU/ml penicillin  and 50 μg/ml streptomycin . The cells were then incubated in a humidified atmosphere containing 5% CO2 at 37 °C. After repeated passage, a new cell line of FDOV1 was successfully established with more than 80 generations. FDOV1 cells have been sent to China General Microbiological Culture Collection Center for preservation (No. 13812) in March 2017.The fresh tissue (1 cm3 cells/100 μl) were seeded in 96-well plates in sextuplicate. Ten days later, 100 μl mixture (CCK-8/medium 1:9) were added into the wells for incubation by 2 hours. We then measured the Optical Density (OD) at 450 nm (OD450) by a microplate reader .Cell proliferation and growth curve was determined by Cell Counting Kit-8 (CCK-8) test. FDOV1 cells (2 × 106 cell) were kept overnight in 3 ml 75% ethanol at − 20 °C. After treatment, the cells were collected and stained with propidium iodide solution for 20 min at 4 °C in darkness. We then assessed the cell cycle by flow cytometer .Resuspended FDOV1 cells  were injected subcutaneously into the dorsal flanks of two mice. Visible tumors were noted in both mice 20 days after transplantation.All the procedures were approved by the Department of Laboratory Animal Science in Fudan University. For in vivo studies, 4–6 weeks old NOD/SCID mice  were maintained under sterile conditions. FDOV1 cells  were used to stain 4-μm sections. The markers used for immunohistochemistry were listed as follows: HNF-1β , Napsin A , Pax-8 , ER  and PR . Microscopic slides were reviewed by a senior gynecology-dedicated pathologist (Prof. Yang).The WES was performed by 3D Medicines Corporation and the specific method was the same as a previous publication .Mutations of the specific genes in FDOV1 cells were validated by Sanger Sequencing. The RNA was extracted by Trizol Methods, then reverse transcription polymerase chain reaction (RT-PCR) was performed as our previous study . The priAdditional file 1:Table S1. The primer of Sanger sequence for specific genes mutation in FDOV1. (DOCX 15 kb)


Monotherapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) still leads to incomplete responses in most EGFR-mutation positive non-small cell lung cancer (NSCLC) patients, often due to acquired resistance through activation of parallel compensatory pathways. We have previously shown that co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3), and Src-yes-associated protein 1 (YAP1) was highly synergistic in vitro and in vivo. In the present study, we treated EGFR-mutation positive cell lines with the combination of osimertinib plus a natural compound, pterostilbene, which has been reported to abrogate Src and STAT3 activation.Methods: Cell viability assays and immunoblotting were performed to reveal the mechanisms of action of pterostilbene, osimertinib and pterostilbene plus osimertinib in five EGFR-mutation positive NSCLC and one triple negative breast cancer (TNBC) cell lines.Results: Osimertinib plus pterostilbene yielded synergistic effects in all EGFR-mutation positive NSCLC cell lines investigated. Surprisingly, pterostilbene alone did not inhibit, nor downregulate Src phosphorylation in the EGFR-mutation positive NSCLC cell lines or the TNBC cell line, MDA-MB-231. However, the double combination of osimertinib plus pterostilbene reversed the osimertinib-induced STAT3, YAP1, and CUB domain-containing protein-1 (CDCP1) phosphorylation and slightly suppressed Src phosphorylation in PC9 and H1975 cells.Conclusion: The results of this study indicate that pterostilbene may be used to abrogate the activated resistance pathways of single osimertinib treatment in EGFR-mutation positive NSCLC. Future studies should focus on in vivo translation and confirmation of these results. OngThe genetic or pharmacologic inhibition of Src family kinases (SFKs) or YAP1 diminishes the phosphorylation of the RTK AXL and the transmembrane protein CUB domain-containing protein-1 (CDCP1) Figure 1). In this study we explored whether pterostilbene inhibits compensatory osimertinib-induced signaling pathways, and if the combination can optimize the upfront therapy of EGFR-mutation positive NSCLC cells.Pterostilbene  is a stilbene of the family of phytoalexin compounds, found in in blueberries and Pterocarpus marsupium (PM) heartwood. It is structurally similar to resveratrol, a compound found in red wine that has comparable antioxidant, anti-inflammatory, and anti-carcinogenic properties. Due to the presence of two methoxyl groups, pterostilbene has increased lipophilic and oral absorption and therefore increased bioavailability compared to resveratrol 2 37°C cell culture incubator and routinely evaluated for mycoplasma contamination.Human lung adenocarcinoma PC9 cells, harboring EGFR exon 19 deletion and 11-18 cells, harboring EGFR exon 21 L858R mutation were provided by F. Hoffmann-La Roche Ltd. , and by Dr. Mayumi Ono, , respectively. EGFR exon 19 deletion positive HCC4006 and HCC827 cells were purchased from the American Type Culture Collection (ATCC). The H1975 cell line, harboring both EGFR exon 21 L858R and resistant T790M mutation as well as the TNBC MBA-MB-231 cell line were purchased from ATCC. All cell lines were maintained in RPMI  1640 supplemented with 1% penicillin/streptomycin/glutamine (Gibco) and 10% fetal bovine serum  in a 5% CO1) was purchased from Amazon.com, Inc. , the second pterostilbene compound (hereafter referred to as pterostilbene2) was bought from Sigma Aldrich/Merck KGaA,  and osimertinib was bought from Selleck Chemicals . Drugs were prepared in dimethylsulfoxide (DMSO) at a concentration of 10-100 mM stock solutions and stored at -20°C. Further dilutions were made in culture medium to final concentration before use. All antibodies used in our study, including dilution and company catalog number can be found in Table 1.The primarily used pterostilbene compound (hereafter referred to as pterostilbene3 (PC9 and H1975) 1.5 x 103 , and incubated for 24h, as previously described 50) determination, MTT viability assays were performed using pterostilbene doses ranging from 0-150 μM. To determine the combined effect of pterostilbene and osimertinib, drug doses in viability assays were as follows: H1975 and HCC4006 cells were treated with osimertinib ranging from 0-90 nM, and pterostilbene ranging from 0-180 μM, or with the combination of both. PC9 and HCC827 cells were treated with osimertinib ranging from 0-120 nM, and pterostilbene ranging from 0-120 μM, or with the combination of both. 11-18 was treated with osimertinib ranging from 0-330 nM, and pterostilbene ranging from 0-120 μM, or with the combination of both. After 72 h of treatment incubation, 0.5 mg/ml of MTT was added to the medium in the wells for 2 h at 37 °C and formazan crystals in viable cells were solubilized with 100 μl DMSO and spectrophotometrically quantified using a microplate reader  at 565 nm of absorbance. Fractional survival was then calculated as percentage to control cells. Data of combined drug effects were subsequently analyzed by the Chou and Talalay method Cells were seeded in 96-well plates at the following densities: 1 x 10PC9 and H75 1.5 x For immunoblotting experiments, 1.5 million cells were seeded in T75 flasks . The next day, PC9, H1975 and MDA-MB-231 cells were either treated with increasing doses of pterostilbene , or with 20 nM osimertinib, 10 μM pterostilbene and a combination of both. Untreated cells received an equivalent dose of vehicle (DMSO). After 24 h cells were washed with cold PBS and re-suspended in ice-cold radioimmunoprecipitation assay (RIPA) buffer  containing protease inhibitor mixture (Roche). Following cell lysis by sonication and centrifugation at 18620x g for 10 min at 4 °C, the resulting supernatant was collected as the total cell lysate. Briefly, the lysates containing 45 μg proteins were electrophoresed on 10% SDS-polyacrylamide gels  and transferred to polyvinylidene difluoride (PVDF) membranes . Membranes were blocked in Odyssey blocking buffer . All target proteins were immunoblotted with appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies. Chemiluminescent (HRP-conjugated) bands were detected in a ChemiDoc MP Imaging System (Bio-Rad laboratories Inc.). β-actin was used as an internal control to confirm equal gel loading. Experiments were performed in biological triplicates with similar results, and representative blots were shown.50) of pterostilbene1 in all cell lines , it could be concluded that single pterostilbene1 treatment was not able to induce anti-proliferative effects in these cell lines, with IC50s ranging from 23.8 to 40.7 μM. However, when combining pterostilbene1 with the third-generation TKI osimertinib , we obtained synergistic results in all EGFR-mutation positive NSCLC cell lines investigated, with a combination index (CI) ranging from 0.63 to 0.70 . These results indicate a synergistic interaction between pterostilbene1 and osimertinib in these cell lines.To confirm the effects of pterostilbene on cell growth, cell viability assays were performed in 5 different EGFR-mutation positive NSCLC cell lines: PC9, H1975, HCC827, HCC4006 and 11-18. After establishing the half maximal inhibitory concentration RTKs, as can be seen in Figure . An increase of ERK, CDCP1, STAT3 and Src phosphorylation was observed upon different concentrations of pterostilbene1 treatment. In contrast, both total and phosphorylated YAP1 were inhibited by pterostilbene1 at higher concentrations. It can also be seen that phosphorylation of Src, YAP1, pCDCP1 and pAXL are correlated, indicating an interrelationship between thse signaling nodes as previously described 1 is opposite from previously published results, in which pterostilbene was reported to be a Src and MET inhibitor in TNBC To determine which signaling nodes are being abrogated by pterostilbene1 in the MBA-MB-231 TNBC cell line was determined. Cells were treated with 0 or 10 μM pterostilbene1 for 24 h, and Src activation was explored. Surprisingly, instead of inhibiting Src phosphorylation, pterostilbene1 caused an induction of Src phosphorylation . To confirm this, we purchased pterostilbene from a different supplier (pterostilbene2), and cell viability experiments were performed to compare the two drugs. In Figure  it can be seen that the two pterostilbene compounds have nearly identical anti-proliferative effects in two EGFR-mutation positive NSCLC cell lines. To verify these results in the TNBC cell line, MBA-MB-231 cells were treated with both pterostilbene compounds. The cell lysates were consequently used for immunoblotting, and again revealed similar mechanisms of action, as can be seen in Figure . Both compounds clearly induce Src phosphorylation.To explore these contradictory results, the activity of pterostilbene1. As we have previously reported, osimertinib alone attenuated ERK activation in both cell lines, but induced CDCP1 and Src activation in PC9 cells, and STAT3, YAP1 and AXL activation in both PC9 and H1975 cells . In contrast, STAT3, YAP1 and AXL phosphorylation are completely abrogated when treated with single pterostilbene1. MET phosphorylation is slightly activated upon single pterostilbene1 treatment in the PC9 cell line. While in both cell lines CDCP1 activation is inhibited compared to baseline levels, Src phosphorylation is not abrogated with single pterostilbene1 treatment. Osimertinib plus pterostilbene1 reversed the osimertinib-induced STAT3 and YAP1 phosphorylation, abolished CDCP1 and AXL activation and decreased Src phosphorylation. In the PC9 cell line, a restoration of MET phosphorylation levels to baseline was also observed with the double combination. The above results indicate that osimertinib treatment activates parallel compensatory pathways, which can be abrogated by combined osimertinib plus pterostilbene treatment.Although in our study pterostilbene was not able to inhibit Src phosphorylation, its effects on YAP1, Src, CDCP1 and AXL phosphorylation in EGFR-mutation positive cells led us to explore the combination of osimertinib plus pterostilbeneEGFR-mutation-positive NSCLC cells co-express RTKs and non-RTKs, especially, Src, YES and FAK, which cannot be inhibited by single EGFR inhibitors, including the third-generation EGFR TKI osimertinib Figure ), the combination of osimertinib plus pterostilbene was synergistic in five-, and downregulated the activation of STAT3, Src-YAP1 and CDCP1 in two EGFR-mutation positive NSCLC cell lines .Pterostilbene is a natural nutrient from blueberries that can be safely used in high doses 28 High MUC1 mRNA expression is correlated with poorer overall survival, and pterostilbene-based inhibition subverted the tumor-microenvironment to an anti-tumoral state by reducing stemness induction of TAMs. Therefore, MUC1 expression levels may predict which patients can benefit from pterostilbene treatment. Another important piece of information is that constitutively active STAT3 increases MUC1 expression levels on both the mRNA and protein level, explaining our synergistic in vitro results.29 Therefore, further investigation in terms of feasibility and in vivo experiments should be carried out.Pterostilbene was shown to modulate tumor-associated macrophages (TAMs), by modulating MUC1.Figure . This study will assess if pterostilbene is an economical but highly hopeful approach to increase the antitumoral activity of EGFR inhibitors in EGFR-mutation positive NSCLC patients, and should clarify whether this combined treatment strategy can be safely incorporated into routine clinical practice.We are currently performing a proof of concept single-center clinical study, based on the model depicted in


Epithelial–mesenchymal plasticity (EMP), encompassing epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET), are considered critical events for cancer metastasis. We investigated chromosomal heterogeneity and chromosomal instability (CIN) profiles of two sister PMC42 breast cancer (BC) cell lines to assess the relationship between their karyotypes and EMP phenotypic plasticity. Karyotyping by GTG banding and exome sequencing were aligned with SWATH quantitative proteomics and existing RNA-sequencing data from the two PMC42 cell lines; the mesenchymal, parental PMC42-ET cell line and the spontaneously epithelially shifted PMC42-LA daughter cell line. These morphologically distinct PMC42 cell lines were also compared with five other BC cell lines  for their expression of EMP and cell surface markers, and stemness and metabolic profiles. The findings suggest that the epithelially shifted cell line has a significantly altered ploidy of chromosomes 3 and 13, which is reflected in their transcriptomic and proteomic expression profiles. Loss of the TGFβR2 gene from chromosome 3 in the epithelial daughter cell line inhibits its EMT induction by TGF-β stimulus. Thus, integrative ‘omics’ characterization established that the PMC42 system is a relevant MET model and provides insights into the regulation of phenotypic plasticity in breast cancer. Breast cancer, a leading cause of cancer death in women, is recognized as a molecularly heterogeneous disease ,2. BreasEpithelial–mesenchymal plasticity (EMP), which encompasses epithelial–mesenchymal transition (EMT) and its reversal , including hybrid states within this spectrum, is considered an important hallmark of cancer that drives metastasis ,18,19. EEMP studies in cancer cell lines have been widely reported. Therein, EMP is either induced by external stimuli, such as hypoxia , TGF-β , EGF 3232, FGF , or by iThe relationships between different levels of regulation  in the intrinsic endogenous plasticity  seen in several EMP systems has not been systematically studied, although each has separately been implicated in EMP regulation. Here, we interrogate and integrate the molecular portrait of PMC42, a unique human breast carcinoma cell line originally established from the pleural effusion of a breast cancer patient in 1983 . OriginaCells  were obtained from the American Type Culture Collection . PMC42-ET and the derivative PMC42-LA cell line were derived from a breast cancer pleural effusion with appropriate institutional ethics clearance  ,42,43,45TM, Thermo Fischer Scientific, Waltham, MA, USA) and antibiotics penicillin and streptomycin , and maintained at 37 °C, 5% CO2, and 95% humidity. Cells were routinely tested and were negative for Mycoplasma.Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/L), L-Glutamine (0.5 g/L) and sodium pyruvate (0.1 g/L) , and supplemented with 10% foetal bovine serum  for 3–4 h. Cells were harvested using trypsin (Corning™ 25053CI) and the pellet was gently treated with hypotonic solution (75 mM KCl) for 40–60 min at 37 °C and fixed in cold methanol/acetic acid (3:1). Two or three drops of suspended cells were applied to glass slides and chromosomes were stained with DAPI and imaged by confocal microscopy . Counting was performed manually in ImageJ. Karyotype assessment via G-band analysis was performed by a commercial genotyping service  and 50 metaphases were analysed per cell line.Genomic DNA was extracted from cells using Bioline Isolate II Genomic DNA kit (Cat: BIO-52067) as per the manufacturer’s instructions. After quantifying the DNA and checking the purity, DNA samples were shipped to GENEWIZ  for whole exome sequencing (WES) and subsequent analysis. Genewiz, Inc.  performed initial quality control assessments and subsequent exome capture using the SureSelectXT HS Target enrichment kit (Agilent). All samples were paired-end multiplex sequenced (2 × 150 base pairs) on the Illumina Hiseq 2500 platform to a median target depth of over 50× WES data have been deposited at NCBI under BioProject ID PRJNA557326.Paired-end reads underwent quality control before alignment to the reference human genome (hg19) using Burrows-Wheeler alignment   and SAMtCRAVAT , a tool specifically tailored to analyze cancer-specific variants , was useTM cDNA Synthesis kit from Bioline. Real time-quantitative PCR (RT-qPCR) was performed using the SYBR Green Master Mix in a ViiA7 Real-Time PCR system (Applied Biosystems) and analysis performed using the QuantstudioTM Real-Time PCR software v1.1 . The primers sequences are listed in Total RNA was extracted from cells using TRIzol (Life Technologies) and subsequent reactions were carried out as per the Bioline Isolate II RNA Micro kit manufacturer’s instructions, and cDNA was synthesized using SensiFASTp < 0.01 were also examined by Ingenuity Pathway Analysis® (IPA) for functional annotation and gene network analysis. The GSVA method from the GSVA R/Bioconductor package was also applied on the gene expression data for the PMC42-ET and PMC42-LA cell lines to score samples against the TGFβ-EMT signature.mRNA transcript abundances for PMC42 cell lines were measured using RNA-seq as previously reported ; data haCells were washed with ice-cold phosphate buffered saline (PBS), and lysed directly in cell lysis buffer containing 4% (w/v) SDS, 10 mM dithiothreitol (DTT), 10 mM Tris-HCl along with Roche compete protease and phosphatase inhibitors . Lysates were sonicated to shear DNA, and protein concentration was quantified using the Pierce™ BCA Protein Assay Kit . On the basis of protein quantifications, each experimental sample was aliquoted into 25 μg samples for processing using the FASP method . DigestiProtein Pilot (V 4.1) software from SCIEX was used for peptide identification. The human protein library was built using the UniProt database  with thCells were lifted with Accutase®  and stained with anti-human CD44-FITC (BD Pharmingen) and anti-human CD24-PB (Exbio) antibodies at manufacturer’s recommended dilutions in 0.1% BSA  diluted in DPBS for 1 h in a rotary shaker at room temperature. Cells were analysed in the presence of propidium iodide (1 µg/mL) using a BD LSRFortessa (BD Biosciences). After doublet discrimination and compensation for spectral overlap, data were analysed by using FlowJo Software (BD Biosciences). For TGFβR2 surface expression, cells were stained with primary antibody  as per manufacture recommended dilutions for 1 h and then with secondary goat antibody for 1 h.TM Delta Surface-150687). During immunocytochemistry, the growth medium was discarded, and cells were washed thrice gently with Dulbecco’s modified PBS . Briefly, cells were fixed in 4% paraformaldehyde ± 0.1% Triton X-100 (depending on the desired permeabilization conditions), rinsed with DPBS, and incubated with primary antibodies at 4 °C overnight. After rinsing again in DPBS, cells were incubated with an appropriate fluorescence-conjugated secondary antibody  were seeded at a density of 20,000 cells per well . The cells were allowed to grow for 24–48 h at 37 °C in 5% CO2, after which the cells were washed and replaced with assay media . The cells were incubated for 1 h at 37 °C in a non-CO2 incubator. Mitochondrial complex inhibitors -phenyl-hydrazone (FCCP) and combined 1 µM rotenone with 1 µM antimycin A) were preloaded in the injection ports. For basal rate measurements, ECAR (Extra Cellular Acidification Rate) and OCR (Oxygen Consumption Rate) measurements were assessed. Experiments were performed in triplicates and the data were normalized by cell number. Collagen -coated Seahorse cell culture plates .ESR1, PGR and ERBB2 gene products, showed substantial variation across the cell lines and revealed two major branches  and the expression of these surface markers was simultaneously assessed by FACS analysis  and the MDA-MB-468 cell line . Compared to MCF-7 and T47D, all other cell lines were mainly constituted by cells with high levels of CD44, and except MDA-MB-468 also showed lower proportions of CD24 expression, consistent with their mesenchymal subgrouping , had a ubgroup; ), had higrouping A,B. +/high/CD24−/low cell populations across PMC42-ET, PMC42-LA, MCF-7 and MDA-MB-468 were also assessed after stimulation of the cells with EGF or TGF-β for 72 h. Both MCF-7 and MDA-MB-468 cell lines exhibited a 10%–20% increase in their CD44high/CD24low proportions after treatment with EGF, but not with TGF-β. PMC42-ET cells, however, could be made potentially stem-enriched with 93%–94% population of cells in CD44high/CD24low state after stimulation with either EGF or TGF-β, which was due to increased CD44 expression , TNFA signalling via NFκb , inflammatory response signature  and hypoxia signature  were observed in PMC42-LA with respect to its parental cell line PMC42-ET using GSEA . Of these, 73 proteins were significantly upregulated, and 61 proteins were significantly downregulated by a factor of 2-fold or more in the epithelial PMC42-LA cells. KEGG pathway analysis indicated that differentially regulated proteins were involved in glycolysis/gluconeogenesis  (p = 0.00073), proteasome  (p = 6.06 × 10−8), protein processing in endoplasmic reticulum (p = 3.06 × 10−6) and carbon metabolism  (p = 0.00039), respectively. The volcano plot that shows the difference in protein levels between PMC42-ET and PMC42-LA also highlights several EMT markers, among which mesenchymal markers, such as VIM and EGFR, were upregulated by 4-fold (p < 2.51 × 10−5) and 2-fold (p < 5.83 × 10−5), respectively, in PMC42-ET cells (p < 0.00066) and 4.6-fold (p < 00093), respectively. IPA also deduced glycolysis (with a z-score of 3.5), aryl hydrocarbon receptor signalling (with a z-score of 3.1) and ILK signalling (with a z-score of 1.6) as significantly upregulated canonical pathways in the epithelial PMC42-LA cell line. Gene network enrichment plot from proteomics analysis indicated a possible role of NFκb complex dysregulation in PMC42-LA cells , including numerical CIN (resulting in aneuploidy) and structural CIN  are inherent in cancer and underpin many of the phenotypic manifestations that contribute to cancer progression. Hence, in order to assess their possible contributions to the spontaneous MET seen in the PMC42 system, we explored whether karyotype differences existed in the PMC42 cell lines using metaphase spreads and karyotypic G-banding.r2 = 0.67 and 0.59, Pearson correlation) , and a negative correlation (r2 = −0.51)  was found between chromosomes 9 and 12, in the PMC42-ET cell line (p < 0.0001)  C. Two ob 0.0001) . Second,p-arm of chromosome 9 is fused with the long arm of chromosome 10, and chromosome 21 has a third copy of its p-arm fused with the long arm of chromosome 7 , TP53, TSC2) and an additional mutated tumour suppressor gene ATM in PMC42-ET only. Eight commonly mutated oncogenes (ARNT (TF), EML4, GNAS, NTRK3 (PK), PER1 (TF), TCL1A (TF), TLX1, TTL) and 2 additional mutated oncogenes TAL1 and IL6ST in PMC42-ET only were also identified for their role in tumorigenesis (SNAI2 (K188N) and SOX3 (R22P) (GSN (R397W), WNT1 (T363K), ITGA4 (R565W) and NID2 (G426E) (ESRP2 (R248S) was found to be mutated in the parental PMC42-ET cell line.WES was performed to probe more deeply the genetic aberrations in the PMC42 system. After applying selective filters for delineating deleterious mutations within exons, we identified 465 SNVs in PMC42-ET and 475 SNVs in PMC42-LA A,B. We cH7, CFTR E. We alsH7, CFTR F. The TPigenesis ; Table 33 (R22P)  and othe (G426E) . In addiThe estimated copy number ratios of chromosomal segments for PMC42-ET relative to PMC42-LA are shown in FOXD2, FOXE3 and the chromosome 3 region containing genes GADL1, RBMS3, TGFBR2 were completely lost from the PMC42-LA cell line. The specific functions of FOXD2 and FOXE3 genes are yet to be determined, however the role of TGFBR2 in EMT induction is well established  compared to the parental MCF-7 cells, which do [p-value 1.6 × 10−5). Therefore, we hypothesised that the gain in chromosome 13 upregulated the expression of various genes that drives the signalling mechanism of ER in PMC42-LA, causing it to be represented as an upstream regulator in our comparative IPA findings. The results show a high degree of concordance between the relative transcript and proteome abundance across the PMC42 cell lines (r2 = 0.736). The significant genomic differences in the PMC42 cell lines led us to ask whether the changes in chromosome content mediated changes in transcriptome and proteome that could influence phenotype determination. The relative transcriptome and proteome abundance for a given chromosome across the two PMC42 cell lines were computed from logosome 13 C. PMC42-which do , , MDA-MB-468  and MDA-MB-231, SUM-159 . The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), as indicators of lactic acid production during glycolysis and mitochondrial respiration during OXPHOS, respectively, were measured . InteresAlthough the requirement of MET in metastasis is somewhat controversial ,73,74,75GSN (R397W), WNT1 (T363K), SNAI2 (K188N), ITGA4 (R565W), SOX3 (R22P) and NID2 (G426E)  . SNAI2-np-value of < 0.001) , as a major attenuated pathway between PMC42 cell lines. Significant assessment from in vitro studies have highlighted the significance of various cytokines, such as TNF-α [Comparison of transcriptome and proteome analyses across PMC42 cell lines, combined with GSEA and IPA, were performed to gain insights into the biological processes dysregulated within our model system. In our transcriptome studies, the expression of forkhead box A1 (FOXA1) gene, which acts to control transcription of estrogen receptor-regulated genes and repress the basallike features of breast cancer cells ,85,86,87< 0.001) . These ras TNF-α ,93 and gas TNF-α ,93,94. Furthermore, our recent published work also investigated the dynamic interconversions observed between the transitional epithelial and mesenchymal subpopulations delineated by EpCAM profiling in the predominantly epithelial PMC42-LA breast cancer cells. The subtleties of this transition vary in proportion of epithelial and mesenchymal phenotypes as determined from single cells clonal propagation. Differences observed in the functional attributes of the single cell-derived clones further explains the stochastic nature and the intrinsic cellular plasticity in PMC42-LA. Interestingly, the implementation of whole exome sequencing across the EpCAM-high and -low subpopulation indicates that observed intrinsic phenotypic plasticity in PMC42-LA was not attributable to chromosomal instability . Moreovep-value < 0.0001). PMC42-ET has the higher chromosome number, consistent with a relative increase of its transcriptome and proteome abundance (In this pattern of karyotypic differences reflected in the genomic copy number alterations across the two cell lines, we observed that the allele fractions had significantly deviated in the PMC42-LA daughter cell line (bundance C, a broabundance . The gaibundance C. Strikibundance C. Thus, bundance . Differehigh/CD24low profile as a reflection of a mesenchymal state [high/CD24low profile as compared to the parental PMC42-ET cells. There are other reports of ambiguity regarding functional aspects of these markers [CD44 and CD24 are considered putative stem cell markers ,97 and eal state ,39,100.  markers , suggestALDH1A3, ALDH3A1 and ALDOC between cell lines. The analysis of metabolic phenotypes emphasises the importance of using bioenergetics profiles to decipher phenotype assessment, rather than drawing conclusions solely from mutational profiling, RNA-seq and/or proteome data [Understanding the mechanistic basis of metabolic alterations and their role in tumorigenesis is currently an area of intense research interest ,105,106.ome data ,108.TGFBR2 gene from the derivative PMC42-LA cell line contributes to a reduction in intrinsic TGFB signalling, and these cells are also refractory to TGF-β-induced EMT, however this pathway did not emerge from GSEA analysis. Comparative RNA-seq also demonstrated the PMC42 system to be an authentic MET model. The inter-data relationships illustrate a high degree of concordance between the relative transcript and proteome abundance and chromosome copy number variations across the PMC42 cell lines and identified putative targets to reverse MET. The novel findings provide mechanistic insights into how genomic instability and karyotypic variations have led to acquisition of new autonomous clonal karyotype and phenotype, which is still related to, but distinct from the parental cell line. However, further metabolomics and epigenetics studies might be more compelling to add into the paradigm to determine implications for mesenchymal to epithelial plasticity changes. Overall, this investigation provides an example of the heterogenous changes that may occur in expanding cancer populations and provides evidence for the levels on which these changes occur to affect their phenotypic properties.In conclusion, the data we have presented herein underscore profound differences in the PMC42 system by allowing for a comprehensive integration of whole exome sequencing, RNA-seq, proteomics and karyotyping data. The identification of novel somatic mutations and indels provide insights into the differences between the two cell lines at genetic scale that potentially drive the phenotypic differences from the genomic level. The loss of


EGFR mutational status as a model for genetic profile. Clonogenic assays of EGFR mutant (n = 6) and wild-type (n = 9) non-small cell lung carcinoma (NSCLC) cell lines were performed independently by two oncologists. Clonogenic survival parameters SF2, SF4, SF6, SF8, mean inactivation dose (MID), D10, D50, α, and β were obtained using the linear quadratic model. The differences in the clonogenic survival parameters between the EGFR mutant and wild-type cell lines were assessed using the Mann–Whitney U test. As a result, for both datasets, the p values for SF2, SF4, D50, α, and α/β were below 0.05, and those for SF2 were lowest. These data indicate that a genetic profile of NSCLC cell lines might be predictive for their radiation response; i.e., EGFR mutant cell lines might be more sensitive to low dose- and low fraction sized-irradiation.In the era of precision medicine, radiotherapy strategies should be determined based on genetic profiles that predict tumor radiosensitivity. Accordingly, pre-clinical research aimed at discovering clinically applicable genetic profiles is needed. However, how a given genetic profile affects cancer cell radiosensitivity is unclear. To address this issue, we performed a pilot in vitro study by utilizing  Radiation therapy is one of the most important treatments for cancer. In the clinical practice of definitive radiation therapy, the doses required for clinical tumor control vary widely even for tumors arising in the same anatomical sites. For example, an integrated analysis of 50 single-institution studies performed by Okunieff et al. revealed that the dose required for 50% clinical tumor control ranged from 21.4 to 90.3 Gy for breast cancer, 50.4 to 83.4 Gy for supraglottic cancer, and 24.3 to 64.4 Gy for cervical cancer .EGFR mutant and wild-type cell lines by non-parametric two-sided Mann–Whitney U test. Correlation of the survival data between datasets was examined by Spearman’s rank order test. Differences were considered statistically significant at p < 0.05. Analyses were performed using Prism8 .Clonogenic survival parameters were compared between


Different cellular uptake, DNA repair rate, and/or increased drug detoxification might be some of the reasons; however, the molecules and signaling pathways responsible for these processes are largely unknown. In the current study, we purified the A2 and B2 from the BLM and tested the cytotoxicities and the molecular mechanisms of each individual compound or in combination with six different cell lines, including a Chinese hamster ovary (CHO) cell line defective in glycosaminoglycan biosynthesis. Our data suggested that glycosaminoglycans might be involved in the cellular uptake of BLMs. Moreover, both BLM and A5 shared similar signaling pathways and are involved in cell cycle and apoptosis in different cancer cell lines.Pingyangmycin is an anticancer drug known as bleomycin A5 (A5), discovered in the Pingyang County of Zhejiang Province of China. Bleomycin (BLM) is a mixture of mainly two compounds (A2 and B2), which is on the World Health Organization’s list of essential medicines. Both BLM and A5 are hydrophilic molecules that depend on transporters or endocytosis receptors to get inside of cells. Once inside, the anticancer activities rely on their abilities to produce DNA breaks, thus leading to cell death. Interestingly, the half maximal inhibitory concentration (IC Both pingyangmycin and bleomycin are used in combination with other antineoplastic agents to treat testicular, ovarian, cervical, head-neck, esophageal, thyroid carcinomas and lymphomas ,2. PingyBLM is a mixture of mainly two components, A2 55%–70%) and B2. The structures of A2, B2, and A5 are shown in 0% and B250 of BLMs vary greatly in different cancer cell lines, ranging from low nM to μM [It is well known that the ICnM to μM . SeveralSince a key target of BLMs is the cellular DNA, it suggests that efficient repair of BLM-induced DNA lesions might be one mechanism explaining the observed difference in cytotoxicity. Indeed, increased DNA repair through prolonged doubling time, evasion of G2/M arrest, and reduced apoptosis are observed , HoweverSaccharomyces cerevisiae, several well studied ABC transporters showed no involvement in the efflux of bleomycin [The increased drug detoxification has been proposed to be the second mechanism responsible for the variable cytotoxicity . The mulleomycin . Thus faA third mechanism suggests that BLM-hydrolase, a thiol protease bleomycin hydrolase (BLH1), can metabolically inactivate BLMs in normal and tumor tissues. However, studies from yeast clearly excluded a role played by this enzyme in bleomycin’s cytotoxicity . More reA fourth and highly relevant mechanism that would largely account for BLM resistance is the limited uptake of the BLMs into cells. It is well known that the cellular plasma membrane limits BLM entry ,21. Thisl-carnitine transporter 2 (hCT2) [l-carnitine transporter 2 (hCT2), which normally transports l-carnitine and polyamine, can also transport the polyamine-containing A5, but not A2 and B2 in cancer cells [Several transporters, such as OCT-1, l-carni2 (hCT2)  and olig2 (hCT2)  are proper cells . More imer cells ,27,28, bProteoglycans are proteins that have one or more covalently linked glycosaminoglycan (GAG) chains. GAGs are linear polysaccharides composed of a hexosamine residue  and an uronic acid residue (glucuronic or iduronic acid), except keratan sulfate, which does not contain uronic acid residues. Moreover, iduronic acid (IdoA) residues are enriched in dermatan sulfate or chondroitin sulfate B but are rare in other types of chondroitin sulfates. The quantity and composition of heparan sulfate/chondroitin sulfate (HS/CS) disaccharide vary, not only from cell to cell, but also from tissue to tissue  because GAGs are the major components of “glycocalyx”, a surface layer of glycans that cover all animal cells. The thickness of the glycocalyx is 50 to 100 times more than that of the cell membrane lipid bilayer ,32. HoweIn the current study, we purified A2 and B2 from BLM and tested BLMs’ cytotoxicities and the underlying molecular mechanisms in six different cell lines, including a unique Chinese hamster ovary cell mutant, defective in GAG biosynthesis (CHO745) , which im/z = 472.18, z = 3; m/z = 707.77, z = 2)   B, which , z = 2) C, which 50 values in the six cell lines were summarized in The cytotoxicity of each pure compound, A2, B2, and A5, the natural mixture of BLM , and the artificial mixtures of A2:A5 (2:1) and B2A5 (1:2) were tested in four cancer and two CHO cell lines. We used human colon cancer cell lines, HCT116, because it does not express hCT2, which is a transporter for pinyangmycin. Therefore, HCT116 has no preference for taking-up A5 over BLM through hCT2 . The humOverall, all BLMs were more toxic to the four cancer cell lines than to the CHO cell lines . Among tIn general, BLMs were more toxic to CHOK1 cells than that of CHO745, which suggested that an inadequate amount of cell surface GAGs in CHO745 made it less susceptible to BLMs’ cytotoxicity, indicating that GAGs might be involved in the BLMs’ cellular uptake.Since A5 was more toxic to all six cell lines tested compared to BLM (A2 + B2), especially to the human lung cancer cell line, A549, and the human colon cancer line, HCT116 , we thenIt is known that BLM induces G2/M cell cycle arrest in cancer cell lines ,39,40. T50 values shown in To this end, both A549 and HCT116 cells were treated for 24 h, either with BLM or A5. Since A5 was more toxic to both A549 and HCT116 cells compared to BLM , we chosSimilar results for G2/M cell cycle arrest were also observed in HCT116 cells, after treating the cells with two different concentrations of BLM or A5 B. At theFurthermore, we examined the effects of BLM and A5 on key proteins, including p21, cdc25C, cyclin B1, Bcl-2, Bax, PARP, involved in DNA damage, cell cycle arrest, and apoptotic pathways, in both A549 and HCT116 cells by Western blotting, which was performed on the whole-cell lysates after the treatments, with either BLM or A5 at two different conditions . p21 and cyclin B1 are indicated in response to DNA damage. The upregulated P21 and downregulated cyclin B1 expressions are associated with cell cycle arrest. Indeed, both BLM and A5 induced p21, resulting in the over-expression and downregulation of the cyclin B1 protein level in both A549 and HCT116 cells at two treatment conditions . cdc25C Bcl-2 and Bax are members of the expanding Bcl-2 family that play key roles in the regulation of apoptosis . Bcl-2 iDecades of research have shown that EGFR forms part of a complex signal transduction network, which is essential for many important cellular processes, including cell proliferation, migration, survival and adhesion . EGFR exWe purified  and systResistance to BLMs’ cytotoxicity and BLM-induced lung fibrosis are two major clinical concerns for both BLM and A5 ,48,49. ICHOK1 cells express cell surface GAGs, whereas CHO745 cells do not ,37. Our 6 cells [It has been reported that cancerous tissues express nearly twice as many GAGs as normal tissues, and both GAG quantity and structures are different for both lethal and non-lethal cancers . Most ma6 cells ,54,55,566 cells  when A54GAGs are well documented endocytosis receptors for different types of molecules and CHOK1 and CHO745 are the cell lines mostly used to demonstrate GAGs as endocytosis receptors . GAGs ar50 data  directed against EGFR, such as gefitinib or erlotinib, are among the first molecular-targeted agents to be approved in the US and other countries, for the treatment of various human cancers with over-expressed or over-activated EGFR. However, cancer patients that have benefited from costly EGFR TKI therapy quickly generate resistance to the drug with 70% known ,59,60 anHuman colorectal cancer cell lines HCT116 and HT29 and human lung cancer cell lines A549 and H1299 were obtained from the type culture collection of the Chinese Academy of Sciences (China); Chinese hamster ovary cell lines CHOK1 and CHO745 were kind gifts from Dr. Jeffrey D. Esko . Chinese hamster ovary (CHO) cell lines (K1 and 745) were described in detail during the past by both Esko  and by uAntibodies for cyclin B1, p21, Bax, EGFR, Bcl-2, PARP, β-Actin, cdc25C, Phospho-EGFR  was from Cell Signaling Technology ; PI/RNase was from BD Biosciences ; BCA protein assay kit was from Beyotime Biotechnology Co. Ltd. . A5 was from Tianjin Tai-he Pharmaceutical Co., Ltd. ; BLM was from Nippon Kayaku Co., Ltd. . All of the other chemicals and reagents were analytical or better grade. Spectramax M5 plate reader was from Molecular Devices ; OLYMPUSCKX41 inverted microscope and cell counting chamber were from Shanghai Qiujing Biochemical Reagents Instrument Co., Ltd. ; Cell culture dishes and 96-well standard plates were from Corning ; 0.22 μm Millipore filter papers were from Whatman ; ZORBAX C18-chromatogram column  and the high performance liquid chromatography (HPLC) system were purchased from Agilent Technologies Co., Ltd. ; the FC500 MPL flow cytometer was from Beckman ; and the mass spectrometer was from Thermo Finnegan .18 . Solvent A was 0.08 M acetic acid (pH 4.3); solvent B was 100% acetonitrile. The gradient used was 5%–20% B in 20 min; the column temperature was 40 °C. The flow rate was 0.8 mL/min, the injection volume was 20 μL, and BLM was monitored at 254 nm. A2 and B2 were collected and then the Thermo Scientific LTQ XL mass spectrometer  with electrospray-ionization was used for examining the purity of A2 and B2 in a positive-ionization mode.BLM hydrochloride injection (15 mg) was dissolved in 250 μL double distilled water, separated and collected by using the Agilent 1260 infinity high performance liquid chromatography (HPLC) system. The HPLC conditions were as followings: chromatographic column C2 and 95% air in the incubator. Cells were kept sub-confluent and media were changed every other day. All cells used were between 3 and 30 passages. DMSO was used to dissolve compounds and the final concentration of DMSO in all cell-related experiments was 0.1%.HCT116 and HT29 cells were maintained in McCoy’s 5A media . H1299 and A549 cells were maintained in RPMI-1640 media as described  , A5, A2, B2, A2A5, B2A5  in 200 μL of complete media, and A2 and B2 accounts for 65% and 35% of the total weight of A2A5 and B2A5, respectively. After 48 h of incubation, 20 μL of resazurin  was added to each well. After 16 h of incubation at 37 °C, the fluorescent signal was monitored by using a Spectramax M5 plate reader at 544 nm excitation wavelength and 595 nm emission wavelength. The relative fluorescence unit (RFU) generated from the assay was proportional to the number of living cells in each well. 4 cells/plate). After 24 h of plating, cells were then treated with the BLM or A5 for 24 h with various doses  at 37 °C with 5% CO2 and 95% air in the incubator, and then harvested by trypsinization, fixed with cold 75% ethanol at 4 °C for 24 h and washed with phosphate buffer saline (PBS) twice. The cell pellet was incubated in a solution containing 500 μL PI/RNase at room temperature in the dark for 30 min. The cells were analyzed by using a flow cytometer. HCT116 and A549 cells were plated on 10 cm diameter plates  or artificial mixtures of A2A5 and B2A5 in six different cell lines. Our data suggested that BLMs were more toxic to four cancer cell lines than to the CHO cell lines. For CHO cell lines, BLMs were more toxic to CHOK1 cells (GAG sufficient) than to cells of CHO745 (GAG deficient), indicating that GAGs might be involved in the BLMs’ cellular uptake. Moreover, we demonstrated that both BLM and A5 share the same molecular signaling pathways in conducting their cytotoxicities based on the cell cycling data, the immunoblot analysis of important proteins involved in the cell cycle, apoptosis, and other processes.


This article describes the effect of the oxidative stress inducers Angiotensin II and 6-hydroxydopamine (6-OHDA) on different cell lines. The levels of expression Angiotensin type 1 and type 2 receptors in different dopaminergic cell lines are shown. The data indicate that treatment with Angiotensin II and 6-OHDA increases the production of reactive oxygen species (ROS) and decreases cell viability. NRF2 is a transcription factor induced by ROS. We provide data that NRF2 overexpression increases cell viability in response to oxidative stress inducers compared to control cells, and that these inducers can, both separately and in combination, enhance the expression of NRF2-regulated genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo1) and Kruppel like factor 9 (Klf9). Interpretation of these data and additional information is presented in the research article “Angiotensin II induces oxidative stress and upregulates neuroprotective signaling from the NRF2 and KLF9 pathway in dopaminergic cells“   We also22.1Dopaminergic Mes23.5 cell line, dopaminergic N27 cell line and SH-SY5Y cell line were cultured as previously described HA–NRF2, kindly donated by Dr. Donna D. Zhang  was used to infect cell lines. Vectors were transfected into HEK 293 T cells with the corresponding viral packaging plasmids. Viruses were collected from the supernatant and used to infect neuronal cell lines in the presence hexadimethrine bromide . Cells were selected with puromycin  starting 72 h after infection. Stable cell lines were established after 2 weeks of selection and were continuously grown in 5 µg/ml of puromycin afterwards.2.2Cell viability was assessed 24 h after the last treatment: cultures were incubated with MTT  for 4 h at 37 °C. Culture supernatant was then carefully removed and the formazan crystals resulting from cellular dehydrogenases were dissolved in acidic isopropanol and quantified at 570 nm using a plate reader  after gentle shaking. The absorbances were expressed as percentage of control.2.3Nrf2, Nqo1, Hmox1 or Klf9, AT1 or AT2 using Actin as housekeeping gene. Primer sequences are for rat Hmox1 F 5′-TCTATCGTGCTCGCATGAAC-3′, R 5′-CCTCTGGCGAAGAAACTCTG-3′; mouse Hmox1 F 5′-CCGCCTTCCTGCTCAACAT-3′, R 5′-CTGACGAAGTGACGCCATCT-3′; rat Nqo1 F 5′-GCCCGGATATTGTAGCTGAA-3′, R 5′- GTGGTGATGGAAAGCAAGGT-3′; rodent Klf9 F 5′-AGTGCATACAGGTGAACGGC-3′, R 5′-AACGGAACTGCTTTTCCCCA-3′; rat Nrf2 F 5′-CCAGCACATCCAGACAGACA-3′, R 5′-ATATCCAGGGCAAGCGACTC-3′; rodent Actb F 5′-TCGTGCGTGACATTAAAGAG-3′, R 5′-TGCCACAGGATTCCATACC-3’; human HMOX1 F 5′-GAGCCTGAATCGAGCAGAAC-3′, R 5′-AGCCTTCTCTGGACACCTGA-3′; human NQO1 F 5′-AAAGGACCCTTCCGGAGTAA-3′, R 5′-CCATCCTTCCAGGATTTGAA-3′; human KLF9 F 5′-GGAAACACGCCTCCGAAAAG-3′, R 5′-GCCGTTCACCTGTATGCACT-3′; human ACTB F 5′-GGACTTCGAGCAAGAGATGG-3′, R 5′-AGCACTGTGTTGGCGTACAG-3′. Total RNA (without reverse transcription) was used as a negative control. Relative mRNA expression was calculated by using comparative cycle threshold (2−ΔΔCt) method and the relative amount of mRNA was presented in the form of fold change over control.Total ribonucleic acid (RNA) was extracted from cell lines using Trizol . Equal amounts of RNA (2 µg) were reverse-transcribed to complementary DNA with Moloney murine leukemia virus reverse transcriptase  following manufacturer׳s instructions. PCR using real-time iCycler PCR platform  in combination with iQ SYBR Green Supermix  were used to assess expression of 2.4L-cysteine  was used as an antioxidant and control for low ROS generation, and hydrogen peroxide  was used as a control for high ROS generation. Fluorescent intensity was expressed as relative units (RU) calculated as the difference from time 0, normalized relative to control at 15 min.Cellular ROS measurement was determined using 2′,7′-dichlorofluorescin-diacetate (DCFDA). Cultured cells were washed and loaded with DCFDA  for 30 min at 37 °C. Basal fluorescence was measured at this time (time = 0) using a plate reader (Tecan Infinite M200) at excitation 485 nm/emission 520 nm. Fluorescence was again measured 15 min, 30 min, 45 min, 60 min, 75 min and 120 min after addition of different treatments. AngII (100 nM) and 6-OHDA (40 µM) were added at the same time in these experiments. N-Acetyl-2.5two groups were analyzed by Mann-Whitney Rank sum test and multiple comparisons were analyzed with one-way ANOVA or two-way ANOVA followed by Holm Sidak test. Differences at p < 0.05 were considered as statistically significant. Statistical analyses and graph design were carried out with SigmaPlot 11.0  except otherwise stated.All data were obtained from at least three separated experiments, with a minimum sample size of four wells per group and per experiment. Data were expressed as percentage of the control group of the same batch  and expressed as a mean±standard error of the mean (SEM). Data from


Wolbachia suppresses the replication of +ssRNA viruses such as dengue and Zika viruses in Aedes aegypti mosquitoes. However, the range of viruses affected by this endosymbiont is yet to be explored. Recently, novel insect-specific viruses (ISVs) have been described from numerous mosquito species and mosquito-derived cell lines. Cell-fusing agent virus (Flaviviridae) and Phasi Charoen-like virus (Bunyaviridae) persistently infect the Ae. aegypti cell line Aag2 which has been used for experimental studies with both the wMel and wMelPop-CLA strains. Wolbachia was found to restrict the replication of CFAV but not the PCLV infection in these lines. Furthermore, an additional Ae. albopictus cell line (RML-12) which contained either wMel or wMelPop-CLA was assessed. While no infectious +ssRNA or dsRNA viruses were detected, a PCLV infection was identified. These observations provide additional evidence to support that insect-specific, +ssRNA viruses can be suppressed in cell culture by Wolbachia but -ssRNA viruses may not. •Wolbachia suppresses CFAV in Aag2 wMel and wMelPop-CLA cell lines.•PCLV persistently infects the RML-12 cell line.•lbWoLA cachia does not suppress PCLV in Aag2 or RML-12 wMel and wMelPop-Cell lines. Interestus line . Furthermaterial . PCLV, aide CFAV .Wolbachia to Aag2 cells impacted the replication of these viruses, analysis undertaken by wMelPop-CLA for the presence of CFAV and PCLV by RT-PCR. It was suggested by both studies that Wolbachia severely restricts the replication of CFAV. However, upon tetracycline treatment of these lines to clear Wolbachia, contradictory results were obtained where one study suggested CFAV replication could recover after the removal of Wolbachia while the other confirmed clearance of the infection. Interestingly, it was demonstrated that Wolbachia had no effect on the insect-specific bunyavirus (PCLV) known to persistently infect these lines  stably infected with wMel and wMelPop-CLA were assessed for CFAV and PCLV replication both prior to and following tetracycline treatments. CFAV replication was not detected in Wolbachia infected cultures nor was the virus able to be recovered upon passage in the highly permissive RNAi-deficient Ae. albopictus (C6/36) cell line. Conversely, PLCV was detected and isolated from both Wolbachia infected cell lines.To determine if the introduction of se lines . To addr22.1Ae. aegypti cell line Aag2 has been shown to be infected with two insect-specific viruses (ISVs)  . To detey RT-PCR A. While 36 cells . Absence36 cells B.Fig. 1E2.2Wolbachia supressed the insect-specific bunyavirus PCLV in co-infected Aag2 cell culture, supernatant obtained from Wolbachia-free Aag2 cell and Aag2 cells infected with wMel or wMelPop-CLA was screened by RT-PCR. In contrast to what was observed with CFAV, PCLV was detected in the Wolbachia-free Aag2 line as well as both Wolbachia infected lines. To further confirm if this was an active viral infection capable of infecting new cells, supernatant harvested from both Wolbachia untreated and tetracycline-treated lines was inoculated onto the permissive C6/36 line for 2 successive passages. PCLV replication was confirmed by RT-PCR in C6/36 supernatant obtained from passage 1 suggesting infectious PCLV was produced in the Aag2 line.To determine if 2.3Wolbachia infected cell lines, Ae. albopictus, Wolbachia-free RML-12 cells as well as RML-12 cells stably infected with wMel and wMelPop-CLA were assessed. PCLV was detected by RT-PCR in supernatant from RML-12 lines regardless of Wolbachia infection  and replication strategies (–ssRNA or dsRNA) in mosquitoes are yet to be widely explored. Recently, ISVs have been isolated in high prevalence from both wild type and laboratory-reared mosquitoes  previously shown to replicate within the parental Aag2 cell line  cells with supernatant harvested from the Aag2 line with or without Wolbachia. CFAV was not detected by anti-dsRNA ELISA from passaged, wMel, wMelPop-CLA or matched tetracycline treated lines. This study and Wolbachia-mediated suppression, Wolbachia densities maintained in the line. However, as it's likely that the CFAV infection was lost prior to both the screening by this study and that of To further assess these interactions between ell line . The repwMel- or wMelPop-CLA-infected Aag2 or RML-12 lines. Consistent with Wolbachia-infected Aag2 cells and in inoculated C6/36 cells.  and vesicular stomatitis virus  was not affected by Wolbachia (wStri) in Ae. albopictus cells while Aedes anphevirus  could be isolated from both wMelPop-CLA and wMelPop-CLATET cell lines. Furthermore, in vivo studies by Wolbachia did not suppress the replication of LACV in co-infected Drosophila melanogaster and more recently Bunyaviridae) was not restricted by Wolbachia in Culex tarsalis. Taken in combination, this provides mounting evidence to suggest differential viral interactions with Wolbachia whereby +ssRNA viruses are susceptible to suppression but –ssRNA viruses may not. However, while Wolbachia restricted CFAV replication, additional investigation should be undertaken in Ae. aegypti mosquitoes to confirm that –ssRNA viruses and +ssRNA viruses which display different tropism and transmission strategies are suppressed by Wolbachia in vivo. The mechanisms underlying viral suppression remain unresolved. Investigations into the interactions of Wolbachia with other virus types may provide information critical to understand the impact of Wolbachia on the mosquito virome as well as mechanisms of viral blocking.Interestingly, this and 4Wolbachia restricts the replication of the +ssRNA virus, CFAV, but not –ssRNA virus, PCLV, in co-infected cell culture. These results support previous studies which have suggested similar interactions  cells were cultured at 28 °C in RPMI 1640 medium (C6/36) or Schneider's Drosophila/Mitsuhashi and Maramorosch 50:50 medium (RML-12) supplemented with 10% foetal bovine serum (FBS) and L-glutamine. Ae. aegypti Aag2, Aag2 wMel and Aag2 wMelPop-CLA cells were cultured in Schneider's/Grace's insect medium (50:50) containing 10% FBS and L-glutamine at 26 °C. Tetracycline treated lines were generated from Aag2, Aag2 wMel and Aag2 wMelPop-CLA by the addition of 10 μg/ml of tetracycline to the maintenance media for 3 successive passages.C6/36 and RML-12  was inoculated onto semi-confluent C6/36 cells and incubated at 28 °C for 5–7 days. Supernatant was harvested and immediately passaged onto fresh C6/36 cells for an additional passage, with the remaining supernatant used for RNA extraction. Monolayers were then fixed using 4% formaldehyde/0.5% Triton X for 4 h at 4 °C. To determine if ISVs were present in other Aedes-derived, Wolbachia-infected lines, RML-12 cells  were assessed (with the exception of tetracycline treatment) by the methods described above. Fixed-cell ELISA was performed using the anti-dsRNA mAb 3G1 as per previously described methods -treatment of Aag2, Aag2  methods . RNA was