MDR1/ABCB1) and BCRP (ABCG2) are known ATP-binding cassette (ABC) drug transporters that mediating MDR. ABCB5 is a close relative to ABCB1, which also mediates MDR. Constitutive activation of the EGF receptor is tightly linked to the development of chemotherapeutic resistance. BetA inhibited P-gp, BCRP, ABCB5 and mutation activated EGFR overexpressing cells with similar efficacy as their drug-sensitive parental counterparts. Furthermore, the mRNA expressions of ABCB1, BCRP, ABCB5 and EGFR were not related to the 50% inhibition concentrations (IC50) for BetA in a panel of 60 cell lines of the National Cancer Institute (NCI), USA. In addition to well-established MDR mechanisms, we attempted to identify other molecular mechanisms that play a role in mediating BetA's cytotoxic activity. For this reason, we performed COMPARE and hierarchical cluster analyses of the transcriptome-wide microarray-based mRNA expression of the NCI cell lines panel. Various genes significantly correlating to BetA's activity were involved in different biological processes, e.g., cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination and proteasome degradation. Immunoblotting and in silico analyses revealed that the inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes. In conclusion, BetA may have therapeutic potential for the treatment of refractory tumors.Betulinic acid (BetA) is a naturally occurring pentacyclic triterpene isolated from the outer bark of white-barked birch trees and many other medicinal plants. Here, we studied betulinic acid's cytotoxic activity against drug-resistant tumor cell lines. P-glycoprotein ( Vitex negundo  is a lupane-type triterpenoid firstly identified and isolated in the 18th century by Johann Tobias Lowitz from the outer bark of white-barked birch trees  are classified into either host factors, in which decreased absorption and distribution with increased metabolism and excretion take place for certain drugs, or cancer cell genetic alterations, in which alternative pathways to evade apoptosis and triggered cell death are activated. Therefore, the design of MDR reversing agents should take into account the factors mentioned above to improve the cancer therapeutic approaches.in vitro and metastasis in vivo  is an ubiquitin E3-ligase cell surface glycoprotein, also known as GP78, that is known to play a role in metastasis, tumor progression and recurrence. AMFR regulates cell motility signaling In the present work, BetA's anticancer activity against drug-resistant tumor cell lines was studied, in which either MDR-conferring ABC-transporters  or mutation-activated EGFR were overexpressed. Furthermore, we investigated the transcriptome-wide mRNA expression profiles of tumor 60 cell lines by COMPARE and hierarchical cluster analyses, to determine multiple other unknown molecular determinants that affect the response of tumor cells to BetA. Using gene-hunting approach, one of the sensitivity determinants genes, AMFR, was selected as a target candidate for BetA. We present novel evidence via molecular docking and immunoblotting analysis that inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes.Drug sensitive CCRF-CEM and multidrug-resistant P-glycoprotein (P-gp)-overexpressing CEM/ADR5000 leukemic cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin . Doxorubicin  was added to maintain overexpression of P-gp  in resistant cells  reduction assay was performed to assess cytotoxicity of BetA in a concentration range of 1010IC50 values) deposited at the NCI website (http://dtp.nci.nih.gov) to identify candidate genes mediate sensitivity and resistance, respectively, to BetA. This gene-hunting approach is based on Pearson's rank correlation test. To obtain COMPARE rankings, a scale index of correlation coefficients  has been generated.We performed COMPARE analysis for a transcriptome-wide search for correlations between gene expressions and BetA response (loghttps://discover.nci.nih.gov/cimminer/), we performed agglomerative hierarchical cluster analysis (WARD method) to cluster the mRNA expression of genes identified by COMPARE analysis and a heatmap was prepared accordingly.Using the CIMMINER program  were treated with varying concentrations of BetA, harvested after 24 h, and washed with PBS. Using M-PER™ mammalian protein extraction buffer (Thermo Fisher Scientific), the entire proteins were extracted from the cell lysates. Then, sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed to isolate the proteins, the proteins were then transferred to polyvinylidene fluoride membranes (Ruti®-PVDF)  for western blotting. Five percent of bovine serum albumin was used to block the membranes and then the membranes were incubated with specific primary antibodies against AMFR   and β-actin  . The blots were probed with horseradish peroxidase-linked IgG secondary antibodies  for 2 h at room temperature. Finally, LuminataTM Classico Western HRP substrate  was added for 5 min in the dark. Alpha Innotech FluorChem Q system  was used for documentation and band analysis.Both sensitive and resistant breast cancer cell lines (10Table 3). Using AutodockTools-1.5.6rc3, the proteins' PDB files were converted to PDBQT format and set as the macromolecule, upon which docking is to be performed. 2D structures of the BetA was constructed and later converted to 3D structures using Corina Online Demo. A grid box was allocated to define docking spaces upon the macromolecule. Energies for each atom type in the ligand were calculated at each grid point using Autogrid 4.2. These calculated energies were later used to predict binding affinity of BetA. Docking was carried out using Autodock 4.2 with 250 runs and 2.5 million evaluations for each cycle via the Lamarckian algorithm. Lowest binding energies were retrieved from the correspondent dlg file and amino acids were analyzed by Autodock Tools. Images were created using Visual Molecular Dynamics VMD.Molecular docking was done in an approach previously reported by us -overexpressing CEM/ADR5000 cells and drug-sensitive parental CCRF-CEM cells using a resazurin assay. No cross-resistance of the CEM/ADR5000 cells was observed  with the log10IC50 values of the NCI cell lines were correlated with standard anticancer drugs . Clusters 5, 6, and 7 contained in its majority of cell lines sensitive to BetA, whereas clusters 1, 2, 3, and 4 contained in its majority resistant ones.The mRNA expression values of all NCI cell lines for the genes listed in Table 10IC50 values for BetA in sensitive NCI cell lines was observed. This prompted us to investigate whether the expression of AMFR in our MDR cells model will be affected when treated with BetA or not. Therefore, we performed western blot analysis after treating breast cancer cell lines with varying concentration of BetA. The results showed that BetA was able to inhibit expression of AMFR in a dose dependent manner . In addition to its ligand AMF. As shown in Table In this study, we investigated the cytotoxic activity of BetA against multidrug-resistant cancer cells and determined the molecular mechanisms associated with either sensitivity or resistance of cancer cells toward BetA. Therefore, we treated cancer cell lines expressing different MDR conferring genes  with BetA. To determine the molecular mechanisms, we mined the NCI's Developmental Therapeutics Program database for the documented screening of BetA with a panel of 60 cancer cell lines. The rationale of this approach was to define genes, whose expressions correlated to the pattern of cellular responsiveness to BetA. The genes assessed by this approach belonged to diverse classes, e.g. oncogenes, tumor suppressor genes, drug resistance mediating transporters, heat shock proteins, telomerase, cytokine receptors, molecules of the cell cycle and apoptotic pathways, DNA repair enzymes, components of cytoskeleton, intracellular signaling molecules, and enzymes of metabolism  transporters  family activates signaling pathways regulating cellular proliferation, angiogenesis initiation, apoptosis inhibition and survival, which subsequently results in increasing tumor masses and chemotherapy refractoriness. In glioblastoma tumors, the common EGFR mutation is in frame deletion of exon 2–7 resulting in ligand-binding domain deletion of the EGFR , signal transduction and transcriptional regulation , protein synthesis , chromatin remodeling , cell adhesion , tumor suppression , ubiquitination and proteasome degradation . All of these mechanisms foster tumor progression.Since the established MDR mechanisms investigated in this study were not involved in resistance toward BetA, we performed a microarray-based transcriptome wide screening of genes using COMPARE analysis, whose mRNA expression correlated with the login vitro and in vivo. The AMF–paclitaxel conjugate inhibited colon and breast cancer cells proliferation more effectively than free paclitaxel. Intra-tumoral injection of the AMF–paclitaxel conjugate also induced more effective tumor regression and increased survival in K1735M1 and B16-F1/paclitaxel resistant mouse melanoma models , inhibitors of CDKs and tumor suppressors . Many tumor cells overexpress CDKs increasing their proliferative capacity. CDK-cyclin complexes tightly monitor the four sequential phases of cell cycle, namely G1, S, G2, and M phase, and dysregulation in any phase results in uncontrolled cell growth  did not hamper BetA activity in tumor cells. Furthermore, microarray-based expression profiling of 60 NCI cell lines revealed that BetA exerts cytotoxic activity toward cancer cells by multiple mechanisms rather than by a single one. Summing up all findings in this study, we can postulate that BetA is a promising candidate with multiple modes of action to treat refractory tumors. Further investigations are needed to assure its efficacy and safety in vitro experiments and wrote the manuscript; NM: performed COMPARE and cluster analysis; YS: provided ABCB5 transfected cell lines; HA-A: designed the bioassays and edited the manuscript; TE: supervised the work, provided the facilities for the study and edited the manuscript. All authors read the manuscript and approved the final version.MS: carried out the HA-A was employed by Steigerwald, Darmstadt, Germany. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neuroblastoma (NB) is the most common and aggressive pediatric cancer, characterized by a remarkable phenotypic diversity and high malignancy. The heterogeneous clinical behavior, ranging from spontaneous remission to fatal metastatic disease, is attributable to NB biology and genetics. Despite major advances in therapies, NB is still associated with a high morbidity and mortality. Thus, novel diagnostic, prognostic, and therapeutic approaches are required, mainly to improve treatment outcomes of high-risk NB patients. Among neuroepithelial cancers, NB is the most studied tumor as far as PPAR ligands are concerned. PPAR ligands are endowed with antitumoral effects, mainly acting on cancer stem cells, and constitute a possible add-on therapy to antiblastic drugs, in particular for NB with unfavourable prognosis. While discussing clinical background, this review will provide a synopsis of the major studies about PPAR expression in NB, focusing on the potential beneficial effects of hypoglycemic drugs, thiazolidinediones and metformin, to reduce the occurrence of relapses as well as tumor regrowth in NB patients. Neuroblastoma (NB) is a tumor of the developing sympathetic nervous system observed in early childhood, which is characterized by a broad spectrum of clinical behaviors, ranging from complete regression to death.NB represents the second most common extracranial malignancy of childhood, accounting for 8 to 10% of all childhood cancers  and for approximately 15% of the pediatric deaths for malignant conditions [The clinical presentation of NB ranges from asymptomatic masses to primary tumors that cause critical illness due to local invasion and/or widely disseminated disease. Most primary NB (65%) usually present in the abdominal region, often in the adrenal medulla. Other common sites of disease include the neck and head (5%), chest (20%), and pelvis (5%) .NB is a disease of the sympaticoadrenal lineage of the neural crest and originates from neuroblasts in the developing peripheral nervous system .In recent years, it has been suggested that NB tumorigenesis is dependent on the presence of cancer stem cells (CSCs), which have been also isolated from NB cell lines , 4. CSCsCellular heterogeneity is a hallmark of NB nodules and the prognosis of these tumors depends on their differentiation levels . InteresBiedler et al. described three cell subtypes, often discernible also in NB cell line cultures, based on cell morphology, biochemical features, and growth patterns : (i) N-tSeveral studies have shown that these cell types derive from a common precursor and are able to bidirectionally differentiate. This bidirectional conversion between well-defined differentiation lineages of the neural crest has been termed “transdifferentiation” .Because the transdifferentiation process is able to also allow the differentiating of malignant CSCs into benign phenotype, a novel concept in cancer biology was introduced: “induction of differentiation” as possible treatment .The cause of NB development is still unclear occurring mostly as sporadic disease but also rare  familial cases were reported –38. OtheTraditional NB treatments include surgery, chemotherapy, radiotherapy, and biotherapy . HoweverUnfortunately, in many cases, by the time of diagnosis, the disease has usually spread already. In these cases, the mainstay treatment is frequently intensive regimens including combinations of high doses of chemotherapeutics  that oft(2) PPARs. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily.PPARs are activated by fatty acids, eicosanoids, other dietary lipids, and their metabolites, or synthetic ligands , which hα, β/δ, and γ) which are encoded by separate genes [There are three PPAR isoforms  or synthetic pharmacological agonists  have been identified [PPAR-entified , and somentified .β/δ is ubiquitously expressed particularly in liver, intestine, kidney, abdominal adipose tissue, skeletal muscle, and macrophages. It regulates energy expenditure, participating in fatty acid oxidation and regulating blood cholesterol concentrations and glucose levels [PPAR-e levels , 54, 59.β/δ agonists are prostacyclin PGI2, oleic acid, and synthetic agents, such as GW501516, GW7842, and GW0742, which attenuate hepatic steatosis [PPAR-teatosis .γ is expressed within adipose tissue, the large intestine, spleen, skeletal muscle, liver, pancreas, endothelial cells, immune cells, various cancer cells, and brain [PPAR-nd brain –63.γ ligands, while synthetic agonists include thiazolidinediones (TZDs), GI262570, GW1929, and GW7845 [γ has been extensively studied as a pharmacological target in several diseases.It regulates energy storage and has a key role in fatty acid metabolism and glucose homeostasis , 64–66, d GW7845 . PPAR-γ γ agonists, and, among them, pioglitazone and rosiglitazone have been approved by FDA for treatment of type II diabetes [TZDs are the best-characterized pharmacological PPAR-diabetes –72.γ mRNA levels [In addition, another oral hypoglycemic drug, metformin, which directly improves insulin action, modulating AMPK activity (a key energy regulator), increases PPAR-A levels , acting A levels .γ, represent promising therapeutic tools as antitumoral agents [γ agonists were reported to inhibit cell growth and to induce apoptosis in several cancer cell lines in vitro and in vivo [Interestingly, it has been suggested to use PPARs as target for cancer treatment, and several PPAR agonists, in particular acting on PPAR-l agents ; PPAR-γ  in vivo –86, incl in vivo , 88.α (mRNA or proteins) at very low level [β/δ expression data are still incomplete [γ is highly expressed in NB cell lines [All three PPARs isoforms have been identified in NB, although human NB cell lines express PPAR-ow level , 89–91, complete , 92. Conll lines , 12 and ll lines , being mll lines , 89.γ than adult rat brain [γ is involved in the regulation of proliferation and differentiation [In addition, it has been documented that embryonic rat brain and neural stem cells have higher concentration of PPAR-at brain , 93. In ntiation .γ expression is correlated to the maturational stage of NB and therefore to NB patients' outcome [γ agonists induce NB cell differentiation, inhibiting proliferation, neurite outgrowth, and reducing N-myc levels [Interestingly, PPAR- outcome , and PPAc levels .γ in NB, evaluating the effects of several natural or synthetic ligands on cellular proliferation, apoptosis, and differentiation , a high-affinity natural ligand of PPAR-NB cells –16, throIn addition, Rodway et al. have found that the inhibition of NB growth induced by 15-deoxy-PGJ2 can be reduced by the presence of serum lysolipids in the culture medium , while Eγ ligands have been also tested to contrast NB cell growth.Synthetic PPAR-Han and coworkers firstly evaluated the effect of the synthetic ligand, GW1929, in the NB cell line LA-N-5, and found that this compound induces cell differentiation and inhibits proliferation .γ activation, as demonstrated by the use of specific antagonists [GW1929 prodifferentiating effect was shown to be dependent on PPAR-agonists .γ protein amount in NB cell lines, but rather to various cellular conditions associated with the receptor function.In 2005, Valentiner et al. tested the effects of four TZDs  in seven NB cell lines  . All the in vivo, in a metastatic xenograft mouse model [The antiproliferative effect of rosiglitazone was confirmed by the same groupse model , althougγ activity, reducing NB cell growth [Ciglitazone was also used in association with 15-deoxy-PGJ2 to overexpress Rb protein and inhibit PPAR-l growth .γ ligands inhibit cell growth in all cell lines, and the sensitivity seems to be more associated with the cell phenotype than with PPAR-γ expression: indeed, N-type cells are more susceptible to treatment than S-type cells, partly because of their higher capability of undergoing apoptosis.Servidei et al. tested 15-deoxy-PGJ2 and rosiglitazone on 8 NB cell lines, with different phenotypes, including N- and S-types . The twoγ-dependent apoptosis in NB-1 cells [+-induced mitochondrial injury reducing ROS production [Many studies have documented that the inhibitory effects of TZDs on neuroblastoma cell growth are partially due to an increase of apoptosis. Indeed, troglitazone induced PPAR--1 cells  and in S-1 cells . In contoduction , 22.γ ligand significantly inhibits cell adhesion and invasiveness and induces apoptosis, more effectively in SK-N-AS than in SH-SY5Y cell lines. The distinct response of the two NB cell lines is likely due to a reduced phosphorylation of PPAR-γ and consequently its increased activity in SK-N-AS cells. Cellai and colleagues also evaluated the in vivo effect of TZDs in NB xenograft models, confirming their previous in vitro observations [Proapoptotic effects of rosiglitazone were also reported . This PPrvations . Indeed rvations .In addition, rosiglitazone induces differentiation, increasing density of dendritic spines in rat primary cortical neurons .Moreover, in neural stem cells (NSC) from adult mammalian brain, pioglitazone and rosiglitazone directly regulate proliferation, differentiation, and migration .γ pathway [Accordingly, Miglio et al. described the effects of pioglitazone on SH-SY5Y NB cells, in which this agonist promotes differentiation and outgrowth of cell processes, in a dose-dependent manner . In 2014 pathway .γ activation contributes to neuronal differentiation [All these observations are in agreement with previous findings indicating that PPAR-ntiation , 94–99.γ activation mainly results in apoptosis promotion in proliferating cells, PPAR-β/δ natural  and synthetic  agonists induce G1 cell cycle arrest, reduce cell migration and invasiveness, and increase neuronal differentiation in SH-NH-5YSY [While PPAR--NH-5YSY .http://www.who.int/topics/clinical_trials/en/).In summary, all these results suggest the possible use of PPAR agonists as novel therapy for NB, but to date clinical trials are not yet underway .Metformin is biguanide with a well-known safety profile, mainly used as oral antidiabetic drug , 101, whr models –110, andIn particular, metformin seems to selectively affect cancer stem cell survival, inhibiting cancer metastases and thus represents a good potential adjuvant agent for chemotherapy (as reviewed by ).However, the molecular mechanisms of action of metformin are still not completely defined, although it seems that the antiproliferative mechanisms induced by this drug are at least partially diverging from those regulating glucose homeostasis. While the latter is mainly dependent on the AMPK activation, the antitumor activity of metformin is mediated by inhibition of AKT/mTOR , the inhibition of TK activity, or the regulation of chloride channels –116.β/δ expression in muscle cells and activity in endothelial cells [γ activation by rosiglitazone in bone marrow progenitor cells [γ and PPAR-α activity in hepatoma cells [α activation role of metformin was identified to increase GLP-1 receptor levels [As far as the effects of metformin on PPAR activity are concerned, several studies were performed but the results are extremely dependent on the receptor subtype and the cells analyzed. For example, metformin increased PPAR-al cells , 118, real cells . On the or cells  and bothma cells . Finallyr levels .However, although demonstrated in several models, the role of the modulation of PPAR expression and/or activity in the antiproliferative effects of metformin in neuroblastoma has not been addressed yet.The effect of metformin on NB was firstly demonstrated by our groups .We reported that the effects of metformin treatment in human SKNBE2 and SH-SY5Y NB cell lines are a significant reduction in the proliferation rate and cell viability, due to inhibition of AKT phosphorylation and an increased cell death, via apoptosis-independent pathways. These effects were more pronounced in SKNBE2, which are less differentiated, highly proliferative cells than SH-SY5Y cells. Notably, metformin effects were different depending on the differentiating stimuli, being abolished by retinoic acid, but were potentiated by overexpression of NDM29, a noncoding RNA affecting NB malignancy, although both conditions were characterized by a neuron-like differentiated phenotype , 123–128 in vitro results are in agreement with those of Kumar and coworkers who evaluated the antitumor activity of metformin against neuroblastoma in vivo [These in vivo . Oral adIn the same year, Vujic et al. successfully used metformin to inhibit cell proliferation and induce apoptosis in NRAS mutant NB cell lines (SK-N-AS and CHP-212), in which NRAS signaling is constitutively active through the PI3K/AKT/mTOR pathway .In 2015, Mouhieddine and colleagues found that metformin reduces proliferation rate, viability, and invasive potential of NB cell lines SH-SY5Y .Interestingly, focusing on metformin effect on stem cell population within a 3D culture model, these authors reported that this drug is able to decrease, but not abolish, cell sphere-forming ability, significantly targeting and reducing cancer stem/progenitor cell population and thus possibly preventing recurrence.Notably, metformin also reduces MMP-2, a protein involved in extracellular matrix degradation, favoring metastasis and cancer progression .http://www.who.int/topics/clinical_trials/en/).Thanks to the highly selective and cytotoxic effects of metformin on NB cells and on stem cell population in particular , these sDespite advances in standard therapeutic protocols, the prognosis of NB has not gained significant progress, especially concerning the rates of metastasis, the incidence of recurrences, and the long-term survival, which are all correlated with the presence of CSCs. in vitro and in vivo studies reported the successful use of PPAR agonists, and in particular of TZDs, in the inhibition of NB cell proliferation and tumor growth, in the induction of cell death, and in the promotion of cell differentiation. These effects are PPAR-dependent, even if some PPAR-independent effects have been also described. Notably, PPAR agonists also reduce the viability of CSCs, the tumor cell subpopulation responsible of NB recurrence. Similar results were obtained with another hypoglycemic drug, metformin, which acts also via the modulation PPAR activity. In the future, it will be important to examine the contribution of PPARs in the antiproliferative activity of metformin and the possible synergistic effect with known or novel selective PPAR agonists.Recently, many groups focused their attention on PPARs, suggesting the use of several PPAR agonists, currently used as hypoglycemic drugs, for NB treatment. Interestingly, many in vitro as well as in vivo are needed, these hypoglycemic drugs may represent realistic new therapeutic approaches to be used as good adjuvant chemotherapeutic agents in NB treatment.Although further studies


Metabolites from marine fungi have hogged the limelight in drug discovery because of their promise as therapeutic agents. A number of metabolites related to marine fungi have been discovered from various sources which are known to possess a range of activities as antibacterial, antiviral and anticancer agents. Although, over a thousand marine fungi based metabolites have already been reported, none of them have reached the market yet which could partly be related to non-comprehensive screening approaches and lack of sustained lead optimization. The origin of these marine fungal metabolites is varied as their habitats have been reported from various sources such as sponge, algae, mangrove derived fungi, and fungi from bottom sediments. The importance of these natural compounds is based on their cytotoxicity and related activities that emanate from the diversity in their chemical structures and functional groups present on them. This review covers the majority of anticancer compounds isolated from marine fungi during 2012–2016 against specific cancer cell lines. Marine fungi are important source of secondary metabolites useful for the drug discovery purposes. Even though marine fungi are less explored in comparison to their terrestrial counterparts, a number of useful hits have been obtained from the drug discovery perspective adding to their importance in the natural product discovery  and (+)-cryptosporin  experiments. Compounds (5) and (6) exhibited considerable growth inhibition against tumor cell lines MCF-7, NCI-H460, SF-268, and HepG-2 with IC50 values <10 μM. The inhibitory effect of compound (5) against MCF-7 cell line was comparable to cytoxicity of cisplatin which was used as a positive control  was evaluated against HeLa, MCF-7, and NCI-H446 cell lines which showed IC50 values of 10.0, 11.0, and 16.0 μM respectively  and the absolute configurations were supported by NOESY, X-ray crystallography, CD and computational methods. Compounds (8–12) displayed cytotoxic activities against A549 cell line with the IC50 values of 20, 16, 19, 17, and 20 μM, respectively, which were moderately higher than the positive control Adriamycin  led to the determination of its chemical structure. Compound (13) displayed weak antiproliferation activities toward K562 and promyelocytic HL-6 cell lines with IC50 values ranging between 25.8 and 44.9 μM , chrysogine (15) and meleagrin  was evaluated against MCF-7, HepG2, NCI-H460, HeLa, DU145, and MDA-MB- 231 cell lines with the IC50 values of 12.0, 7.0, 10.0, 10.0, 8.0, and 8.0 μg/mL respectively. The cytotoxicity of compound (15) was moderate against SW1990 cell line with an IC50 value of 20.0 μg/mL, whereas compound (16) exhibited potent cytotoxicity against DU145 cell line with an IC50 value of 5.0 μg/mL. It also showed moderate cytotoxicity toward HepG2, NCIH460, HeLa, and MDA-MB-231 cell lines with IC50 values of 12.0, 22.0, 20.0, and 11.0 μg/mL respectively  were isolated from the Antarctic deep-sea fungus Penicillium sp. PR19 N-. The cytotoxicity studies of compounds (17) and (18) against HL-60 cells were evaluated which gave IC50 values of 45.8, 28.3 μM respectively. The inhibitory concentrations of compounds (17) and (18) against A-549 cells were found to have IC50 values of 82.8, 5.2 μM respectively , aspilactonols A–F  and epiaspinonediol (27) were isolated together with five known polyketides (S)-2-(2′-hydroxyethyl)-4-methyl-γ-butyrolactone (28), dihydroaspyrone (29), aspinotriol A (30), aspinotriol B (31) and chaetoquadrin F (32) from Aspergillus sp. 16-02-1, which was collected from a deep-sea sediment at a Lau Basin hydrothermal vent in the southwest of the Pacific Ocean. NMR, mass and CD spectroscopy techniques were used to determine the chemical structure and assign the absolute configuration of these novel molecules. Compounds (19-32) exhibited significant cytotoxic activities with inhibitory rate (IR%) values at 100 μg/mL between 10 and 79% against human cancer cell lines K562, HL-60, HeLa, and BGC-823  and a known polyketide  was modest against HL-60 and A549 cell lines with IC50 values of 11.8 and 12.2 μM respectively  and B  displayed G2/M cell cycle arrest of human lung carcinoma cells  inhibited proliferation and migration of human hepatoma SMMC-7721 cells , ergosterol peroxide (45), -5α,6α-epoxy-3β-hydroxyergosta-22-ene-7-one (46), and cerebroside A-D  and 10-epi-sclerotiamide (52) were the secondary metabolites of the deep-sea fungus Aspergillus westerdijkiae DFFSCS013, which showed excellent cytotoxicity against K562 cell line with IC50 of 44 and 53 μM respectively , T988A (55) and gliocladines C–D  were isolated  was evaluated against SF-268 MCF-7 NCI-H460 HepG-2 cell lines, which gave IC50 values of 0.46, 0.233, 1.15, and 0.91 μM respectively. Similarly, the cytotoxicity of compound (54) against SF-268 MCF-7 NCI-H460 HepG-2 gave IC50 values of 0.59, 0.25, 1.31, 1.29 μM respectively. Compounds (55–57) were slightly less cytotoxic against SF-268, MCF-7, NCI-H460, and HepG-2 with IC50 values ranging in between 0.91 and 17, 7 μM respectively. The positive control cisplatin, exhibited cytotoxicity against SF-268, MCF-7, NCI-H460, and HepG-2 cell lines with IC50 values of 4.7, 3.9, 2.9, and 2.4 μM respectively  were isolated from the deep-sea fungus Penicillium sp. in China  and (59) were determined against MCF-7 cells lines, which gave IC50 values of 7.44 and 28.4 μM respectively. The IC50 of compound (58) against A549 cells was found to be 32.5 μM  and varioloid B  and a known compound insulicolide A (63) whose chemical structures are given in Figure 50 values between 1.95 and 6.35 μM  was obtained from which physcion (64) was isolated  induced apoptosis in HeLa cells and its effect on the expressions of p53, p21, Bax, Bcl-2, caspase-9, and caspase-3 proteins were investigated. The western blot analysis revealed that physcion (64) induces cell apoptosis through down-regulation of Bcl-2 expression, up-regulation of Bax expression, and activation the caspase-3 pathway. Additionally, physcion (64) also induced the formation of reactive oxygen species (ROS) in HeLa cells  , which was isolated from the surface of a marine red alga Lomentaria catenata, collected at Guryongpo, NamGu, PoHang in the Republic of Korea. Neoechinulin A (65) has shown the cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction in HeLa cells was investigated by the expressions of p53, p21, Bax, Bcl-2, Caspase 9, and Caspase 3 proteins. Western blot analysis revealed that neoechinulin A (65) could induce cell apoptosis through down-regulation of Bcl-2 expression, up-regulation of Bax expression and activation the caspase-3 pathway  together with tetranorlabdane diterpenoid derivative (68), wentilactones A (42) and B (43) were isolated. The cytotoxicity of these compounds  was moderate against HeLa, HepG2, MCF-7, MDA-MB-231, NCI-H460, SMMC-7721 and SW1990 tumor cell lines. Wentilactone B (43) was the most potent among the tested compounds (IC50 = 17 μM)  and brocazine G (70) were obtained from mangrove-derived fungus Penicillium brocae MA-231  showed moderate activity against A2780 cells (IC50 59 μM) while compound (70) showed strong activity against A2780 and A2780 CisR cell with the IC50 values of 664 and 661 nM respectively, which were much better than that of the positive control cisplatin, which gave IC50 values of 1.67 and 12.63 μM respectively , and electronic CD experiment. Compound (71) exhibited cytotoxicity against MMQ and GH3 cell lines with the IC50 values of 5.2 and 13.0 μM respectively  together with spreussomerin K (74), preussomerin H (75), preussomerin G (76), and preussomerin F (77) as their metabolites  and (73) were determined by single-crystal X-ray diffraction techniques. Compounds (72) and (73) were the first chlorinated compounds in the preussomerins family, which showed potent in vitro cytotoxicity against A549 and MCF-7 human cancer cell lines with IC50 values ranging from 5.9 to 8.9 μM. Compounds (74–77) exhibited significant bioactivity against A549, HepG2, and MCF-7 human cancer cell lines with the IC50 values of 2.5–9.4 μM  approach, spirobrocazines C (78), pestalotioprolides D-F (79–81) were isolated from mangrove derived endophytic fungus Pestalotiopsis microspore  collected from Douala, Cameroon. Co-culture of P. microspora with Streptomyces lividans resulted in roughly ten-fold enhancement in the production accumulation of compounds (80) and (81) compared to axenic fungal control. Their chemical structures were determined by the analysis of NMR and mass spectroscopy data. The cytotoxicity of these compounds (78-81) was significant against murine lymphoma cell line L5178Y, which showed IC50 values of 0.7, 5.6, 3.4, and 3.9 μM respectively. Compound (80) was potent against the human ovarian cancer cell line A2780 with an IC50 value of 1.2 μM  and ilicicolin H (83) were isolated from Campylocarpon sp. HDN13-307  and ilicicolin H (83) were cytotoxic against HeLa cell with the IC50 values of 8.8 and 4.7 μM respectively , altersolanols A, B, N (85-87), and alterporriol E (88) were isolated from white bean solid culture media of the endophytic fungus, Stemphylium globuliferum, collected from the Egyptian mangrove plant Avicennia marina , altersolanol A (85), B (86), and alterporriol E (88) exhibited toxicity against L5178Y mouse lymphoma cell line with the IC50 values of 3.4, 2.5, 3.7, and 6.9 μM  also exhibited potent cytotoxicity against L5178Y mouse lymphoma cell line with IC50 values in the low micromolar range  showed cytotoxic activity against 34 human cancer cell lines in vitro, with mean IC50 (IC70) values of 0.005 μg/mL (0.024 μg/ml) respectively  has been studied in detail, which has shown that it is a kinase inhibitor which induces cell death by apoptosis through caspase dependent pathway. Altersolanol A (85) inhibited a variety of kinases, which suggested that the kinase inhibition might be the mechanism for the cytotoxic activity , penitrems A, C, F (92–94), and 3β-hydroxy- 4β-desoxypaxilline (95) whose chemical structures are shown in Figure (89-94) were cytotoxic against the human A-549 cell lines with IC50 values of 11.5, 6.3, 9.2, 8.4, 8.0, 8.2, and 4.6 μM, while compounds  showed cytotoxicity against the human HL-60 cell lines with IC50 values of 9.6, 5.0,7.0, 4.7, 3.3, and 2.6 μM respectively. Adriamycin, a positive control, exhibited cytotoxicity with the IC50 values of 0.30 and 0.06 μM against A-549 and HL-60 cell lines respectively  displayed cytotoxicity against the mouse lymphoma cell line L5178Y activity with an IC50 value of 4.10 μM  were isolated from the culture broth of a mangrove-derived endophytic fungus, Rhytidhysteron rufulum, which was obtained from Thai Bruguiera gymnorrhiza  was further confirmed by single-crystal X-ray diffraction analysis. Compounds (97–100) displayed cytotoxicity against Kato-3 cell lines with the IC50 values ranging from 16.0 to 23.3 μM, while rhytidchromones A and C were active against MCF-7 cells with the IC50 values of 19.3 and 17.7 μM respectively  was cytotoxic against several cell lines with the IC50 values of 10.1 μM (MDA-MB-435), 12.5 μM (HepG2), 11.9 μM (HCT-116), 13.31 μM (A549), and 39.74 μM (THP1) respectively  in the South China Sea was the source of beauvericin (102), a cyclic peptide  was potent in the growth inhibition of KB and KBv200 cells with the IC50 values of 5.76 and 5.34 μM. Further analysis of beauvericin (102) activity was done, which showed that it induced apoptosis through the decrease of reactive oxygen species generation, loss of mitochondrial membrane potential, release of cytochrome C, activation of Caspase-9 and -3, and cleavage of PARP and did not regulate Bcl-2 or Bax expression - benzoate  and (104) as shown in Figure (103) was weak against Tca8113 and MG-63 cells with the IC50 values of 26 and 35 μM respectively. The positive control, taxol, gave the IC50 values of 46 and 10 nM with Tca8113 and MG-63 cell lines respectively. The IC50 value of compound (104) on Tca8113 and normal liver cell line WRL-68 was 10 and 58 μM respectively. Compound (104) also showed anti-tumor effect on MG-63 cells with an IC50 value of 55 nM. Compound (104) was also tested against nude mice, which showed significant inhibition of tumor growth of human osteosarcoma . Compound (105) exhibited activities against K562, SGC-7901, and BEL-7402 cell lines with the IC50 values of 9.2, 15.7, and 25.4 μg/mL respectively. Paclitaxel was used as the positive control, which displayed the IC50 values of 5.1 μg/mL against K562, 1.6 μg/mL against SGC-7901 and 6.3 μg/mL against BEL-7402 cel lines respectively , pestalotiopene A (107), cytosporone E (108) were obtained from the endophytic fungus Acremonium strictum, isolated from the mangrove tree Rhizophora apiculata  showed moderate cytotoxic activity against human cisplatin-sensitive  and resistant A2780 cell lines   and G (110) as shown in Figure (109) and G (110) showed significant cytotoxicity against Raji cancer cell line with an IC50 value of 2 μg/mL , which was obtained from a branch of Bruguiera gymnoihiza (Linn.) growing on the coastal salt marsh of the South China Sea was the source of compounds  and beauvericin (102) as shown in Figure (111) and (102) ranged from moderate to strong against MCF-7, A549, HeLa, and KB cell lines with the IC50 values of 4.98 and 2.02 (MCF-7), 1.95 and 0.82 (A549), 0.68 and 1.14 (HeLa) and 1.50 and 1.10 μM (KB) respectively. The inhibitory activity of compound (112) was weak against these tumor cell lines  as shown in Figure (113). Nigerasterols A and B , which represent the first 5,9-epidioxy-sterol compounds of marine origin were evaluated for cytotoxicity. Nigerasterol B (114) displayed potent activity against the tumor cell line HL60 with an IC50 value of 1.50 μM, while nigerasterol A (113) displayed stronger activity with an IC50 value of 0.30 μM. Both compounds (113) and (114) displayed potent activities against A549 cell line with the IC50 values of 1.82 and 5.41 μM respectively  exhibited cytotoxicity against KB and KBV200 cells with the IC50 values of 6 and 10 μg/mL, respectively . Its chemical structure was determined on the basis of spectroscopic data. Compound (116) inhibited the growth of HEp-2 and HepG2 cells with the IC50 values of 9 and 16 μM respectively  led to the production of two metabolites: compound (117) and compound (118) as shown in Figure (117) showed inhibitory activity toward MCF-7 and HL-60 cancer cell lines with the IC50 values of 4.4 and 3.4 μM, respectively. The cytotoxicity of compound (118) was promising against HL-60 cell line with an IC50 value of 0.6 μM  as shown in Figure (119) was cytotoxic against KB and KBV200 cells with the IC50 values 5 and 10 μg/mL respectively  produced pullularin A (120), pullularin C (121), verticillin D (122) and pullularins E and F  as shown in Figure (120–124) were cytotoxic against the mouse lymphoma cells (L5178Y) with the EC50 values between 0.1 and 6.7 μg/mL -7-methoxy-6-methyl-13H)-isobenzofuranone (H-isobenz(125–127) were isolated from the marine fungus Penicillium sp. 303 cultured from sea water samples obtained from Zhanjiang Mangrove National Nature Reserve in Guangdong Province, China  and (126) showed moderate cytotoxic activities against a panel of cancer cell lines including MDA-MB-435, HepG2, HCT-116 and A549 cell lines. Compound (127) showed potent cytotoxic activity with IC50 values of 7.13 μM against MDA-MB-435  was isolated from mangrove endophytic fungus No·Gx-3a in the South China Sea  was obtained from mangrove sediment of Shankou in Hainan, China, which led to the production of a new xanthone derivative (129) as shown in Figure 50 values of 8 and 9 μg/mL  was elucidated by detailed spectroscopic analysis and absolute configuration determination. Compound (130) showed potent inhibition growth of MCF-7, SW1990, and SMMC7721 tumor cell lines with the IC50 values of 4, 5, and 7 μg/mL respectively  was isolated from mangrove endophytic fungus No.5094 which was collected in the South China Sea as shown in Figure (131) showed strong inhibitory activity with KB and KBv200 cell lines having the LD50 values of 5.5 and 10.2 μM respectively  as shown in Figure 132) showed moderate cytotoxicity against MCF-7, NCI-H460 and SF-268 cell lines with the IC50 values 83.9, 13.5, and 25.3 μg/ mL respectively , pseurotin D (134), alkaloids fumitremorgin C (135), and 12,13-dihydroxy fumitremorgin C  which was obtained from the sediments collected on the northeast coast of Brazil. Pseurotin A (133), pseurotin D (134), fumitremorgin C (135), and 12,13-dihydroxy-fumitremorgin C (136) showed toxicity against HCT-116 cell line with the IC50 values 72.0, 85.0, 15.1, and 4.5 μM , tryptoquivaline U (138), and fiscalin B (139) were isolated from Neosartorya fischeri which was obtained from marine mud in the intertidal zone of Hainan Province of China  toward apoptosis of HL-60 cells were done which showed the IC50 values of 82.3, 90.0, and 8.8 μM respectively . Their chemical structures were deduced using NMR and mass spectrometry analysis. Penipacids A (140) and E (141) inhibited RKO cell growth with the IC50 values of 8.4 and 9.7 μM while compound (142) was cytotoxic against HeLa cell line with an IC50 value of 6.6 μM, which was better than the positive control fluorouracil  and a known polyketide compound (144) were isolated  and (144) showed weak cytotoxicity against HepG2 cell line with the IC50 values of 9.9 and 9.7 μM respectively  as shown in Figure (145) was cytotoxic against SK-MEL-5 human melanoma cells with an IC50 value of 0.9 μM  as shown in Figure (146) showed inhibitory activity against three human tumor cell lines; SF-268, MCF-7, and NCI-H460 with the IC50 values of 7.1, 6.6, and 7.4 μM respectively , libertellenone A (148) and diaporthein B (149) whose structures are depicted in Figure (147) showed only mild antiproliferative activity with the IC50 values between 25.6 and 46.0 μM, whereas, libertellenone A (148) and diaporthein B (149), revealed potent antiproliferative activities with IC50 values ranging from 4.4 to 20.0 μM, compared to cisplatin (IC50 = 1.5–9.2 μM)  and compound (151) as shown in Figure (150) and (151) showed cytotoxicity against K562, A549, Huh-7, H1975, HL60, HeLa, and MOLT-4 cell lines with the IC50 values ranging between 1.1 and 47.4 μM. Compound (150) showed cytotoxicity against K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa, and MOLT-4 cell lines, with IC50 values of 10.5, 13.7, 10.9, 19.1, 11.1, 47.4, and 11.8 μM respectively while compound (151) was cytotoxic against K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa MOLT-4 cell lines with IC50 values of 6.2, 1.1, >50, 14.2, 18.5, 3.4, 18.8, 6.2, 3.2, and 4.1 μM respectively. The positive control trichostatin A was cytotoxic to the same cell lines with the IC50 values of 0.24, 0.05, 0.09, 0.10, 0.08, 0.06, 0.09, 0.09, 0.11, and 0.03 μM respectively  which was collected from the coastal forest soil at Samaersarn island, Chonburi Province, Thailand was the source of aszonapyrone A (152), 13-oxofumitremorgin B (153), sartorypyrone A (154) and sartorypyrone B (155) as shown in Figure (152), 13-oxofumitremorgin B (153), sartorypyrone A (154) and sartorypyrone B (155) were evaluated for their ability to inhibit the growth of MCF-7, NCI-H460, and A375-C5 cell lines. The cytotoxicity results displayed that, among the meroditerpenes tested, aszonapyrone A (152) was the most potent compound showing strong growth inhibitory activity with GI50 = 13.6, 11.6 and 10.2 μM for MCF- 7, NCI-H460 and A375-C5 cell lines respectively. Sartorypyrone B (155) was also potent in growth inhibition, however, it was less active than aszonapyrone A (154) having the GI50 values 17.8, 20.5, and 25.0 μM for MCF-7, NCI-H460 and A375- C5 cell lines respectively. Another compound 13-oxofumitremorgin B (153) exhibited only weak inhibitory activity against all the three cell lines  as shown in Figure (156) exhibited in vitro moderate cytotoxicity toward HepG-2 and Caski human tumor cell lines with the IC50 values of 9.31 and 12.40 μg/mL respectively. Compound (157) also displayed selectivity against HepG-2 and Caski human tumor cell lines with the IC50 values of 2.91 and 10.20 μg/mL respectively  was obtained from the Red Sea sponge, Xestospongia sp. which was collected from deep waters of Sharm Obhur, Jeddah, Saudi Arabia  showed antitumor effect against both Ehrlich ascites carcinoma and lymphocytic leukemia (LD50 5.0 μM each)  whose chemical structures are shown in Figure (159) and (160) were assigned using a combination of experimental circular dichroism (CD) investigation and quantum chemical CD calculations. The skeleton of marilines is unusual and its biosynthesis was suggested to require uncommon biochemical reactions in fungal secondary metabolism. Both enantiomers, marilines A1 (159) and A2 (160) inhibited human leukocyte elastase (HLE) with an IC50 value of 0.86 μM  as shown in Figure (161) was cytotoxic against MCF-7 and SMMC-7721 cell lines with the IC50 values of 16.6 and 18.2 μmol/L  and C (163) as shown in Figure (162) and C (163) were selective against melanoma cells B-16 and human gastric cancer cells SGC-7901 with the IC50 values of 0.29, 0.44 and 0.06, 0.72 μM respectively. The control experiments with normal mammary epithelial cells M10 at the same concentration did not show significant toxicity  was isolated from marine-derived fungus Penicillium chrysogenum HGQ6 which was obtained from Lianyungang sea mud sample  was active against BGC823 cell line with an IC50 value of 0.19 mg/mL, which was lower than that of adriamycin with an IC50 value of 0.06 mg/mL , epiremisporine B1 (166) and isoconiochaetone C (167) as shown in Figure (165) exhibited cytotoxicity against K562, HL-60, with the IC50 values of 69.0 and 62.9 μg/mL. Similarly epiremisporine B1 (166) exhibited cytotoxicity against K562, HL-60 cell lines with the IC50 values of 53.1 and 54.7 μg/mL respectively while the percent inhibition rate for isoconiochaetone C (167) were 20.4 and 26.0 at 100 μg/ mL against K562 and HL-60 cell lines respectively  a novel alkaloid with a unique spiro skeleton was isolated from a marine-derived fungus Penicillium citrinum  showed toxicity against A-375, SPC-A1, and HGC-27 cancer cell lines with IC50 values of 20.1, 28.6 and 29.4 μM respectively. Morphological evaluation, apoptosis rate analysis, Western blot and real-time quantitative PCR (RT-qPCR) results showed that penicitrinine A could significantly induce A-375 cell apoptosis by decreasing the expression of Bcl-2 and increasing the expression of Bax. Additionally, anti-metastatic effects of penicitrinine A in A-375 cells by wound healing assay, trans-well assay, Western blot and RT-qPCR were also investigated. These results showed penicitrinine A significantly suppressed metastatic activity of A-375 cells by regulating the expression of MMP-9 and its specific inhibitor TIMP-1  showed moderate activity against PC3 and HCT116 cell line with the IC50 values of 30.4 and 39.2 μM respectively  as shown in Figure (170) was cytotoxic to human carcinoma U937 and MOLT4 cell lines with the IC50 values of 6.4 and 6.2 μM respectively  were produced from marine-derived fungus Aspergillus aculeatus  and (172) were cytotoxic to HL-60, K562, and A-549 cell lines with the IC50 values in the range of 5.4–6.1 μM  and diorcinol E (174)  was moderately cytotoxic against HeLa and K562 cell lines with the IC50 values of 31.5 and 48.9 μM respectively while compound (174) showed cytotoxicity against only HeLa cell line with the IC50 value 36.5 μM  which was obtained from the inner tissue of a pulmonate false limpet Siphonaria sp. that was collected from rock surfaces in the intertidal zone of Moora Park, Shorncliffe, Queensland, Australia. Chaunolidone A (175) was found to be a selective and potent inhibitor of human non-small cell lung carcinoma cell NCI-H460 with the IC50 value 0.09 μM  Figure  were pro(176) and a known compound oxaphenalenone  and (177) on HL-60 cells under the same conditions were determined to be 95.2 μg/ mL (313.2 μM) and 14.0 μg/ mL (56.9 μM). Compounds (176) and (177) also weakly inhibited the K562 cells with inhibition rate (IR) % values of 20.8 and 28.1% at 100 μg/mL (328.9 μM for 176 and 406.5 μM for 177). The positive control 5-fluorouracil inhibited K562 cells with an IR% of 40.3% at 100 μg/mL (796.2 μM)  was produced from a neomycin-resistant mutant of the marine-derived fungus Penicillium purpurogenum G59  was selective against the HeLa cells with an inhibition rate (IR%) of 39.4% at 100 μg/mL which was similar to that of the positive control 5-fluorouracil (IR% of 41.4% at 100 μg/mL against HeLa cells)  was cytotoxic against A375 and SW-620 cell lines with IC50 values of 11.7 and 22.6 μM  showed significant cytotoxicity against A375 cell lines with the IC50 value of 5.7 μM  showed toxicity aginst human gastric cancer cell line SGC-7901 with an IC50 value of 0.29 μM and against hepatic cancer cells BEL-7404 with an IC50 value of 0.31 μM  was cytotoxic against human nasopharyngeal cancer cell lines CNE-1 and 2 with the inhibitory concentration (IC50) values of 1.32 and 0.56 μM respectively  and (+)-6-O-demethylpestalotiopsin C  and (184) were cytotoxic against HL-60 and K562 with the IC50 values 6.9 and 12.3 μM respectively  and a known compound diaporthin B (187) as shown in  were effective in growth inhibition of KB and KBv200 cell lines with the IC50 values of 3.51, 2.34 μg/mL, 20.74, 14.47 μg/mL, and 3.86, 6.52 μg/mL respectively  caused S-phase accumulation and cell death in HeLa cells, suggesting response to DNA double strand breaks  analysis and Mosher's method. The compound (189) showed toxicity against K562, HL-60, BGC-823, HeLa, and MCF-7 cell lines with IC50 values of 60.8, 16.7, 73.8, 75.4, and 59.2 μM  and neoaspergillic acid (191)  exhibited toxicity against Jurkat, K562, U937, and Raji cell lines with the IC50 values of 31.6, 50.1, 42.6, and 54.9 μM respectively. Compound (190) was poorly active against Jurkat cell lines with an IC50 value of 60.2 μM . The active components of this fungus were isolated which resulted in the identification of a novel naphthochromene-2,7-dione skeleton. Compound (192) acts by topoisomerase II inhibition similar to adriamycin activity. Further experiments with BEL-7402 cells showed that (192) reduced cancer growth via a caspase dependent pathway  as shown in Figure 50 value 63 μM after 24 h incubation. Further experiments showed that compound (193) inhibited the protein and gene expressions of MMP-2,-9 in MCF-7 human breast cancer cells by altering MAPK signaling pathway  and oxalicumone A  and (195) exhibited cytotoxicity against eight cell lines  with the IC50 values of <10 μM respectively  is an anthraquinone compound which was obtained from the marine mangrove fungus Nigrospora sp. No. 1403 as shown in Figure in vitro cytotoxicity of deoxybostrycin against MDA-MB-435, HepG2, and HCT-116 cancer cell lines were determined with the IC50 values of 3.1, 29.9, and 5.6 μM respectively  and auranomide C (201) were isolated from the marine-derived fungus Penicillium aurantiogriseum  were elucidated by using spectroscopic methods such as IR, high-resolution mass spectroscopy and two-dimensional NMR spectroscopy. Auranomides A-C (197-199) exhibited moderate cytotoxic activity against K562, ACHN, HEPG2, and A549 cell lines. Auranomide B (199) displayed the best activity among them with an IC50 value of 0.097 μmol/mL against HEPG2 cells  Figure  were isoAs discussed before in different sections in this review article, a total of 199 compounds isolated from marine fungi have shown considerable promise as cytotoxic agents with potential to be developed as anticancer agents in recent years. About half of these compounds have been known to be isoloated from terrestrial or other natural sources previously but they have been reported to be isolated from the marine soruces for the first time. The Supplementary Table 84, 88, 116, 119, 129, 131, 192, and 196) that would enable binding to DNA and RNA, cancer targets that involve nucleic acid recognition should be probed. For example, chromosomal DNA ends in humans, which are rich in guanines, have been shown to form a unique four stranded structures called G-quadruplexes. Both in vitro and in vivo studies have shown the formation of these non-canonical structures which use assembly of four guanines , hydrogen bonded in a Hoogsteen fashion . Furthermore, topoisomerases are enzymes that remove supercoiling in DNA during the replication process and repairs strand breaks  have already shown topoisomerase inhibitory function. Stalling topoisomerase functions by its stabilization with small molecules is another target for anticancer therapy. Since several of these molecules possess structural features that would enable binding to nucleic acids, a screen that targets all forms of nucleic acid structures should be done. This would not only identify the lead compounds for cancer therapy but would also result in identifying the compound that could be of potential use in antibacterial and antiviral therapy.Additionally, since several of these metabolites possess structural features , Bcl-2 downregulation was established as one factor that led to the apoptosis. Do these metabolites function by upregulation of tumor suppressor proteins such as p53 and Bax? The curiosities can be answered only when all protein targets are screened for binding; at least for the ones whose cellular functioning is fairly understood. This would usher a new beginning in the development of natural product based small molecules and possibly identify structural motifs that target specific regions in the protein binding pockets. Such leads can then be used to launch structure activity relationship programs to improve the potency of these leads whose inhibitory concentrations are mostly in the micromolar range. Overall, the discoveries presented in this review highlights many structural classes including some new skeletons that these metabolites produce, which have potential to be developed as clinically useful anticancer drugs. However, in the absence of more detailed studies that focuses on deciphering the cellular events that lead to cell death, their true potential as an anticancer compound might remain to be under-appreciated.In addition to this, there are many new discoveries that could have protein targets within the cancer cells. In fact, the majority of FDA approved anticancer drugs target proteins such as cyclin dependent kinases and histone deacytylase. Could these proteins targets be the potential mechanism by which these metabolites induce cytotoxicity? For some of the compounds (Marine life has been the source of several clinically useful drugs. The findings covered in this review highlight the discoveries of many new natural small molecules, some of them with novel skeletons, that have anticancer activity against a variety of cancer cell lines. The anticancer activity of these compounds is varied with inhibitory concentrations ranging from low to high micromolar concentrations. Some of these metabolites have inhibitory concentrations comparable or better than some of the currently used anticancer drugs. Clearly, these leads have not been explored in detail to determine the actual cellular targets that result in the cytotoxicity and that has been an area whose complete exploration may result in a paradigm shift in the current drug discovery efforts. However, other parallel efforts are needed to facilitate and accentuate marine based drug discovery. One such need is setting-up national and international centers for culture collection since many of the new metabolites reported here have been collected from harsh and hostile environments where the human reach is not easily achievable. This would also help not just in retaining these precious cultures but also in allowing wider reach of these metabolites to specialized groups. A major impediment in marine based drug discovery and, in general, natural product based drug discovery has been the lack of centers that foster programs at the interface of chemistry and biology. Clearly, such specialized centers that have expertise both in chemistry and biology could help in realizing true properties of these metabolites. Morever, lack of complete taxonomy details of the new species and bureaucratic difficulties in the implementation of Nagoya protocol hinder smooth access of knowledge and resources. Therefore, international agreements that clearly address these problems and seek solutions to it, could also greatly help in the smooth exchange of resources. Another important improvement in the area would be developing sustainable biochemical production processes of the screening hits as demonstrated in the case of anticancer compounds Scopularide A and B (Yu et al., SD, VP, and NR reviewed the contents critically. VP and NR drew chemical structures and assisted in the preparation of Supplementary Table The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers. Breast cancer is the most common invasive cancer in women and the leading cause of cancer deaths in females . ImportaIn the last few years several studies have demonstrated that cancer cells produce and release increased numbers of membranous vesicles into the extracellular environment compared to normal cells ,6,7,8. TCancer-derived EVs are in general heterogeneous, but can be divided into two main classes based on their mode of biogenesis and size . These aHere, we use high-throughput sequencing to determine the complete small RNA content in EVs derived from nine breast cancer cell lines. By employing this comprehensive strategy, we have identified common signatures in the breast cancer cell line-derived EVs that are distinct from the intracellular small RNA expression pattern in the originating cells. This difference is mainly due to high contents of non-miRNA small RNAs in EVs, especially a highly abundant small RNA derived from the 28S rRNA. We find, with some exceptions, that the intracellular miRNA expression pattern is mostly reflected in the EVs. Interestingly, the oncogenic miRNA miR-21, which expression is highly associated with breast cancer, is retained in the breast cancer cell lines. This finding supports selectivity in the sorting and secretion of certain miRNA species. Finally, small RNA signatures show that EVs from breast cancer cell lines cluster together, and can be separated from EVs derived from other cancer cell types. This is an important observation that support that small RNAs within cancer-derived EVs may be suitable as breast cancer-specific biomarkers.2 and cultured in either Dulbecco´s Modified Eagle's Medium (DMEM) or Roswell Park Memorial Institute Medium (RPMI) supplemented with 10% FBS.Human cancer cell lines MA-11, MDA-MB-231 (ATCC #HTB-26™), Hs578T (ATCC #HTB-126™), AU565 (ATCC #CRL-2351™), HCC1428 (ATCC #CRL-2327™), MCF7 (ATCC #HTB-22™), HCC1187 (ATCC #CRL-2322™), DU4475 (ATCC #HTB-123™), HCC1569 (ATCC #CRL-2330™), DLD1 (ATCC #CCL-221™), HPAF-II (ATCC #CRL-1997™) and LNCaP (ATCC #CRL-1740™), were maintained at 37°C in 5% COwww.atcc.org). The human MA-11 breast carcinoma cell line, established from bone marrow micrometastases of a patient with breast cancer [All cancer cell lines, except MA-11, were obtained from the American Type Culture Collection  to DMEM or RPMI. Conditioned media was harvested and centrifuged for 30 min at 2,000 g and 4°C to remove detached cells and cell debris. The supernatant was transferred to a new tube and 7.5 ml of Total Exosome Isolation reagent was added and the mixture was incubated over night at 2°C. After incubation, the samples were centrifuged for 1h at 10,000 g. The supernatant was discarded and the pellets consisting of extracellular vesicles were resuspended in 200 μl PBS.EVs were isolated from cell culture media using the Total Exosome Isolation reagent according to the protocol (Invitrogen/Life Technologies), publication number MAN000694. Conditioned media was prepared from approximately 1×10The vesicle size distributions were measured by photon correlation spectroscopy using a Submicron Particle-sizer . To avoid possible interference caused by dust particles, test tubes were pre-rinsed with distilled water and bath-sonicated for 10 min. In addition, all sample preparations were performed in a laminar airflow bench. The vesicle samples were diluted with filtered (0.2 μm Milipore filters) distilled water to provide appropriate count intensity (approx. 250–350 kHz) and measured in three parallels (run time 10 min at 23°C). Both Gaussian and Nicomp algorithms were fitted to the experimental data to find the distribution that best describes the vesicle population. As the fit error was found to be smaller than 1.5, and the residual error was smaller than 10, Nicomp distribution was selected. The volume-weighted distribution was used to determine the mean diameter and polydispersity index (PI) of all samples.Cells and isolated EVs were lysed in RIPA buffer  supplemented with Complete Ultra Mini EDTA-free protease inhibitor mixture  and HaltTM phosphatase inhibitor cocktail (Thermo Fisher Scientific). 30 μg protein extracts were resolved on a SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with antibodies recognizing CD63  or actin . The blots were developed using IRDye800CW-conjugated goat anti-mouse secondary antibody  and the Odyssey IR Imaging system.Small RNA from EVs was isolated using the Total Exosome RNA and Protein isolation Kit according to the protocol, including “Enriching for Small RNAs” step (Invitrogen/Life Technologies), publication number MAN0006962.Total RNA from the cultured cells was isolated using the TRIzol reagent (Invitrogen/ Life Technologies) and enriched for small RNAs using the PureLink™ miRNA Isolation Kit (Invitrogen/Life Technologies).RNA quality and concentration were assessed with the Agilent 2100 Bioanalyzer (Thermo Scientific). Total RNA and RNA after small RNA enrichment was analyzed using RNA 6000 Nano Kit (Agilent) and Small RNA Kit (Agilent), respectively.20 ng of EV RNA and 120 ng cell RNA, both enriched for small RNA, was used as the starting input for RNA-Seq library preparation. The preparation of the cDNA library was done according to the protocol from Applied Biosystems SOLiD™ small RNA library preparation from Invitrogen. The cDNA fragments were barcoded in the PCR amplification step to enable simultaneous sequencing of different samples in a single run. The cDNAs underwent 18 cycles of PCR using barcoded primers. The PCR products were purified using PureLink™ PCR Micro Kit (Invitrogen) and analyzed for size and concentration on Agilent 2100 Bioanalyzer using DNA HS chips. Equal molar amount of each barcoded sample were pooled together in one library, which subsequently were used in emulsion PCR to a total concentration of 0.5 pM. Approximately 1 billion enriched beads were deposited on a full glass slide for SOLiD 5500xl sequencing. Cell RNA from Hs578T and AU565 was previously sequenced on SOLiD4 , SRA accAll bioinformatics analysis was performed with the CLC Genomics workbench 7.0. Raw reads were first subjected to color space adaptor trimming. Further, reads above 29 nt and below 15 nt were discarded and tags with less than 10 copies were removed. All samples were normalized by linear total count scaling, and tags with expression below 100 reads per million (RPM) in all samples were removed.For mapping, the human genome with annotation was downloaded from the Ensembl genome database (GRCh37.74) . MappingHierarchal clustering analyses were performed with custom setting for CLC Genomics workbench .2O) was included for each primer set. The LightCycler® 96 was used for quantification, and the ΔΔCq-method was used to calculate fold change using miR-23 as internal reference. MiR-23 was validated as an internal reference by a bioinformatic approach using the SOLiD small RNA-Seq data. Here, small RNAs with a p-value (EV versus cell line) higher than 0.9 and expression above 1000 read per million (RPM) in both EV and cell lines were filtered for. This resulted in a list of seven small RNAs. MiR-23 was selected as it had the lowest standard deviation out of the miRNAs with a miRNA specific sequence identity.0.2 ng (per replicate) of small RNA enriched RNA were used for small RNA expression quantification using the miRCURY LNA™ Universal RT PCR system  according to the manufacturer recommendations. All experiments were done in triplicates, and a no template control , Basal B , or Basal A , assumed to reflect the most common subtypes, and the heterogeneity of breast cancer. Although breast tumours are classified in six subtypes , only three different subtypes of cancer cell lines are identified based on gene expression; Luminal, Basal A and Basal B. Even though a significant discrepancy of subtypes between primary tumours and cell lines exist, comparison have shown that cell lines mirror both the genomic heterogeneity and the recurrent genome copy number abnormalities found in primary tumours. The sizes of isolated EVs ranged from 17 to 438 nm, with some variations in the relative distribution pattern among the different cell lines (data not shown). Western blot analyses confirmed that the isolated EVs from Hs578T were enriched for the tetraspanin CD63, a well-known exosomal marker protein . We geneFollowing adaptor trimming, size selection, and sampling count, 48,824,105 cellular reads and 23,131,082 EV-derived reads mapped to the human genome (GRCh37.74). Of note, whereas 57% of the reads derived from EV RNA mapped to only a single location in the genome (unique mapping), only 37% of cellular reads mapped uniquely. However, as small RNAs transcribed and processed from non-unique genomic loci might be as important as those deriving from unique loci in terms of both biological functions and cancer biomarkers, no filter was used to exclude sequences based on their mapping position. This generated a list of 222,932 tags (unique sequencing reads) for the breast cancer cell lines and their corresponding EVs. For differential expression analysis and further comparison studies, only tags with expression higher than 100 reads per million (RPM) in at least one sample were included. This resulted in a total of 6,375 tags . The seqTo compare the small RNA transcriptome of EVs and cells, we performed hierarchical cluster analysis . Based oAs hierarchical cluster analyses clearly indicated that the small RNA content in EVs and cells differed substantially, we further investigated the origin of the small RNA species from both compartments. Approximately one third of the reads generated from the cell (27%) and EV (30%) fractions mapped to loci encoding miRNAs . HoweverTo gain more insight into what makes EVs unique in their small RNA content, we examined the twenty most abundant small RNA species in the EVs . InteresWe then went on to analyze the correlation between all EV-derived and cellular small RNA species in each individual cell line. The cells divided into two groups based on the correlation; those that displayed moderate correlation, and those that displayed weak correlation . Not surWe observed that some miRNAs were retained in the cells, including miR-21-5p and miR-29b-3p . Fifty dTo explore if the small RNA signatures of the EVs from the breast cancer cell lines are unique from other cancer EVs, we deep-sequenced the small RNA content of EVs derived from five non-breast cancer cell lines. These include LNCaP (prostate cancer), DLD1 , HPAF-II, (pancreas cancer) and two melanoma-derived cell lines (MM1and MM5). A total of 29,037,189 filtered reads were generated representing 191,210 unique small RNA sequences. Mapping of the sequencing reads to the human genome showed that 28% of the small RNA reads mapped to genes encoding miRNAs, which is similar to what was observed for the breast cancer cell lines . HoweverHigh throughput sequencing has during the last few years broadened the concept of small regulatory RNAs, and novel classes have been discovered with potential biological functions. There are several reports that miRNAs and other small RNA species are incorporated into cell-secreted vesicles, and it is now generally accepted that EVs can act as mediators of intercellular communication ,58,59,60The small RNA content of EVs is dominated by srRNAs that appear specifically secreted from the cells. Most prominent among these are a series of srRNAs that map to the exact 5’end of the 28S rRNA, but which display size variations in their 3’ ends. In small RNA profiling studies, srRNAs have often been ignored as unspecific degradation artifacts that have been generated during for instance library preparations . HoweverSwitching our focus to the miRNAs, we find that miRNAs in EVs released by a particular cell line, largely reflect the miRNA expression pattern within the cells. However, for three of the cell lines very low amount of miRNAs was detected in the EVs even though the overall intracellular level of miRNAs was equal to the other cell lines. This indicates that these cell lines retain their cellular miRNAs. The molecular basis for this phenomenon is unclear, but they may lack key protein components that are required for the incorporation of miRNAs into vesicles that are destined for secretion. Even though our results show that the intracellular miRNAs are largely reflected in the EVs, we find specific miRNAs that are not equally distributed. Previous studies have indeed shown that miRNAs are not randomly incorporated into exosomes ,70,71, aFinally, we find that the miRNA content in EVs derived from eight out of nine breast cancer cell lines generate a specific signature that make them distinct from EVs secreted by other cancer cell lines. This is an important observation that indicates that profiling of miRNAs in EVs isolated from an individual, can contribute in the diagnosis of breast cancer. Taken together, our data argue strongly that small RNA sequences are specifically produced and packaged into EVs. Recently, two papers have demonstrated that tumor-derived exosomes play key roles in the generation of pre-metastatic niches in specific organs ,19. HereS1 FigExtracellular vesicles (EV), whole cell extract (WCE).(TIF)Click here for additional data file.S2 FigLog2 fold change expression of seven small RNAs  in four cell lines  were correlated to the small RNA-Seq data.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


The goal is to provide data that can improve our ability to robustly and reproducibly use human pluripotent stem cells to understand development and disease.The use of induced pluripotent stem cells (iPSC) derived from independent patients and sources holds considerable promise to improve the understanding of development and disease. However, optimized use of iPSC depends on our ability to develop methods to efficiently qualify cell lines and protocols, monitor genetic stability, and evaluate self-renewal and differentiation potential. To accomplish these goals, 57 stem cell lines from 10 laboratories were differentiated to 7 different states, resulting in 248 analyzed samples. Cell lines were differentiated and characterized at a central laboratory using standardized cell culture methodologies, protocols, and metadata descriptors. Stem cell and derived differentiated lines were characterized using RNA-seq, miRNA-seq, copy number arrays, DNA methylation arrays, flow cytometry, and molecular histology. All materials, including raw data, metadata, analysis and processing code, and methodological and provenance documentation are publicly available for re-use and interactive exploration at  While methods of somatic cell reprogramming are being continually improved5, prior to transplantation of iPSC or their differentiated derivatives, extensive characterization of each line must be performed to ensure normal growth characteristics, pluripotentiality, genetic stability and lack of immunoreactivity. For comparison purposes, human embryonic stem cells (hESC) have been considered to be the functional, genetic and epigenetic ‘gold standard’ in this field6. Such analyses are also needed to ensure the reproducible use of existing iPSC lines for laboratory research purposes.The development of methods to transform adult somatic cells into induced pluripotent stem cells (iPSC) have lead to new opportunities for disease modeling and clinical translationin vivo and in vitro differentiation potential. To provide an unbiased evaluation of PSC stability and quality, we recently characterized 57 of these PSC lines using a broad range of phenotypic and molecular omics assays7. A single characterization core laboratory was used to ensure sufficient standardization of these methods. After these characterizations, 57 cell lines were differentiated into 7 different states, including 3 germ layers and embryoid bodies, and subsequently characterized using genomic assays  (The Progenitor Cell Biology Consortium (PCBC) of the National Heart, Lung and Blood Institute was established to investigate and optimize methods for reprogramming and differentiation of iPSC as a precursor to transplantation studies with these lines. The 64 stem cell lines (58 iPSC and 6 hESC) selected were obtained from a diverse set of somatic cell of origins, gene reprogramming combinations, culturing methods  and reprogramming vectors for both distinct and common genetic donors. These lines were sent to a centralized core laboratory for cell culturing, mycoplasma testing, growth characterization, flow cytometry analysis, and icroRNA) .https://www.synapse.org/pcbc) (Data Citation 1), a publicly available online collaborative research platform that provides data annotation, documentation, and file provenance8. Specifically, we deposited metadata, in vitro and in vivo differentiation, qPCR, RNA-seq, miRNA-seq, copy number variation, and DNA methylation data, and processed results from both low- and high-throughput analyses. An interactive browser was developed for querying, filtering, analyzing, and visualizing the genomics data. For users looking to reprocess the raw data, we provide annotations for querying and automatically downloading all raw and intermediate data files. We are also using the portal to distribute insights and results of the analysis as they become available. The data provided is available for unrestricted reuse. We encourage other researchers and members of the public to download and critically analyze this resource.To ensure the comparability of all identifiable covariates, we developed descriptive metadata standards, including ontology defined controlled vocabularies in addition to consistent quality control metrics and data analysis methods. All relevant documentation and data has been deposited in Synapse  established standard protocols for sample collection, handling, and analysis (syn2512369) and the PCBC Bioinformatics Core established standard data processing techniques for the multi-omic data as well as computational quality control. The details of experimental methods for the handling and processing of the stem cells used here have been previously described9 available through the National Center for Biomedical Ontology’s (NCBO) BioPortal10. When ontology terms were not sufficiently defined in their source ontologies, new terms were defined and requested from ontology editors. For example, cell type terms from the Cell Ontology such as ‘endothelial stem cell’ were added to describe the cell line type, and cell terms from UBERON were added to describe the cell line’s tissue of origin.The PCBC Bioinformatics Committee Working Group coordinated the development and implementation of a PCBC cell line characterization metadata standard (syn2767699) for the initially donated cell lines. Through an iterative process, cell line characterization metadata terms were defined and mapped to Open Biological and Biomedical Ontologies (OBO) Foundry ontologiesin vitro genetic and experimental characterization data of the line (such as karyotype status), and resubmitted to the originating lab for confirmation. Metadata fields have also been added to facilitate sample comparisons in downstream analyses (see the Usage Notes).The collected metadata includes detailed information on each associated cell line collected from the submitting investigators including cell of origin, method of reprogramming, reprogramming gene combination, donor sex, ethnicity and disease status (syn2767694). Metadata information was initially provided by the originating laboratory, and was subsequently augmented with Multiple institutions contributed cell lines used in this study, all of which were generated using IRB-approved protocols from the initiating institution. Approval letters or designation of non-human subjects research (for some made with waste products) were received from all institutional IRB. Since no identifying information on the lines was provided to the C4, this study was performed under an Embryonic Stem Cell Research Oversight Committee (ESCRO) approval, as it was not considered human subjects research, and under the IRB at Cincinnati Children's Hospital Research Center.et al.11, and samples were taken at 5, 15, and 30 days after differentiation. The ectoderm differentiation protocol (syn2512373) was adapted from Chambers et al.12. The definitive endoderm differentiation protocol (syn2512372) was adapted from D’Amour et al.13. Cells were additionally differentiated in embryoid body (EB) cultures for 17 days. On day 7, they were grown out from the EB using the embryoid body differentiation protocol (syn2512370). After differentiation, cells were harvested for RNA and DNA extraction.In brief, hESC and iPSC lines were cultured (using protocols syn2724700 and syn2724705) and stored (using protocol syn2724707). Each stem cell line was also evaluated in a teratoma assay (protocol syn3103753) and karyotyped. Reports and images for the teratoma assays (syn2882774) and karyotyping (syn2679104) are available. The lines were then differentiated into germ layer states: ectoderm, definitive endoderm, and mesoderm. The mesoderm differentiation protocol (syn2512371) was adapted from Zhang In brief, gene and microRNA expression were assessed by Illumina HiSeq 2,000 sequencing, and methylation was assessed with the Illumina HumanMethylation450 BeadChip. Two assays were used for copy number variation analysis: 21 cell lines were assayed with the Illumina CytoSNP-850K BeadChip, and 29 cell lines were assayed with the Illumina HD HumanOMNI-Quad BeadChip platform. For validation of gene expression, thirty-seven samples were analyzed using a TaqMan Low Density Array (TLDA)  that interogates a panel of stem cell and pluripotency marker genes (syn3107327).Each assay was processed through one or more reproducible pipelines. Details of the processing for each file is stored in Synapse as part of a provenance record for the file, which links back to code, input files, and parameters. Prior to processing, samples with quality control issues (described in the subsequent assay processing descriptions) were excluded.19. For AltAnalyze analysis, unique putative novel exons were determined from all Tophat junction alignments in AltAnalyze version 2.0.9  and analyzed for associated exon-read coverage using the BedTools function BAMtoBED20, along with all AltAnalyze predicted exons (Ensembl 72 and UCSC annotated mRNAs). The resulting exon and junction BED files for AltAnalyze were used as input for downstream statistical and visualization analyses  in AltAnalyze, using indicated stringency options for transcription, exon and reciprocal junction analyses (syn3105745). These analyses include reciprocal junction Percent Spliced In (PSI) analysis, in which the ratio of junction read counts was calculated for each evaluated exon-exon junction compared to the total number of junction read counts for all genomic overlapping TopHat detected exon-exon junctions (known and novel). Samples without sufficient junction read-depth (>4 reads) for each evaluated splicing event were not considered for statistical analyses. For splicing visualization, coverage plots were produced from the Broad’s IGV Sashimi-Plot function21.All samples were evaluated for a variety of quality control metrics including alignment percentage, proportion of exonic reads, and distribution of reads at the 5′ and 3′ ends of transcripts using the Cincinnati Children’s Medical Center DNA sequencing core automated pipeline. Samples with quality control issues  were flagged in the metadata and were not included in downstream analyses. FASTQ files syn1773112) were aligned to the human genome build GRCh37 and University of California Santa Cruz (UCSC) transcriptome ref. 73112 wer22 using the human miRBase 20.0 (ref. 24 (syn3355992) using the human miRBase 21.0  and were not used in downstream analyses. miRNA expression was quantified with mirExpress v2.1.40.0 ref.  (syn22471.0 ref.  (syn618525 (syn2233188). 15 samples were removed due to poor intensity identified by minfiQC. The remaining samples were quantile normalized and inspected using principal components analysis.DNA-methylation arrays were normalized with the minfi R packageThe plug-in cnvPartition (v3.2.0) for GenomeStudio was used to identify CNVs from the SNP arrays. For this software, the default settings were used, with the exception of a minimum loss of heterozygosity was changed to 5 Mb and minimum number of SNPs changed to 10. Microarray copy number final reports were generated (syn2679103).27 (syn2390898).SNPs from the SNP arrays were augmented with SNPs called from RNA-Seq for quality control purposes. SNPs were called from from RNA-Seq using SamTools 0.1.18 ref.  and subshttp://www.synapse.org/pcbc (Data Citation 1). All computationally derived files also include provenance, a historical record of how, when, and by whom they were generated, which provides the ability to understand the origin of the results, reproduce them, apply the same procedures to new data, and attribute the work performed. The main Project page includes details about the PCBC, overviews of the available data and analyses, and links to main sections of content. Many individual files and folders have their own Wiki content as the equivalent of a ‘README’ file, providing context and detailed information directly with the content. In addition to data files, the analysis code and workflows for all associated files has been included where appropriate. Analysis scripts are also provided through provenance records tracked with individual files. The code and scripts are hosted in Github  and are available for reuse under the MIT license. Synapse accession numbers (in the form synXXXX) are listed with specific files and folders directly in the manuscript. Each record also has an associated Document Object Identifier (DOI) (in the form http://doi.org/10.7303/synXXXXX) that can be used for citation. Each assay  has an associated Folder that contains all raw data files (https://creativecommons.org/publicdomain/zero/1.0/), meaning they can be used for any purpose without restriction. See the C4 data page (https://www.synapse.org/#!Synapse:syn1773109/wiki/218833) for further information.All associated data described in this manuscript are available for download at ta files . Each fo28 This tool is freely available (with a Synapse account) at https://www.synapse.org/#!Synapse:syn1773109/wiki/63531.To aid in interactive analyses of this data, a data exploration tool has been developed to facilitate gene-level and cluster visualization as well as functional enrichment analyses using ToppGeneThe PCBC cell line metadata standard containing over 100 terms is available (syn2767699). Term definitions and URLs for term sources are included. In brief, the top level of terms in each controlled vocabulary (CV) include: PCBC cell line name, originating laboratory ID, C4 cell line ID, host species, cell line type, somatic cell type, iPSC cell type of origin, tissue of origin, reprogramming vector type, induced reprogramming gene combination, culture conditions, donor age, donor life stage, race, ethnicity, sex, disease, donor phenotype, reprogramming laboratory, and PCBC primary investigator.Each PCBC cell line term has been defined and linked to an OBO Foundry ontology term, where available. Ontologies include the NCBI Taxonomy CV, Cell Line Ontology, Cell Ontology, SO: Sequence Types and Features Ontology, NCI Thesaurus, HsapDv, PATO, the Disease Ontology and the Human Phenotype Ontology. The metadata characterization of the lines that are undergoing or completed characterization are available in Synapse (syn2767694). Complete metadata, including specifics about the assays  for each assay are also available: mRNA (syn3156503), miRNA (syn3219876), and methylation (syn3156828).All assay-related files have a minimum set of annotations to facilitate programmatic access. First, all assay files have a unique identifier (UID) for the specific assay . This UID matches up to the records in the assay metadata tables described above and are used to automate the annotation of the individual files. Other annotations are manually added at the time of upload into Synapse. The `dataType` annotation notes which assay the data file comes from: mRNA, miRNA, methylation. The `fileType` annotation denotes the type of file . Many files also have a file sub-type . These annotations can be used to query for specific sets of files, as described below in the Usage Notes section. This provides the abiliity to easily query (using the Synapse clients) for files for a specific cell line, reprogramming vector, or any other attribute described in the metadata tables. Files for a specific cell line across assays can also be identified (using the dataType annotation) or for a specific part of the pipeline process . Example queries are provided below in the Usage Notes section.Experimental protocols used in the processing of C4 cell lines are available for public use (syn2512369). These include protocols for handling, storing, and differentiating stem cells. Furthermore, 5 hub sites of the PCBC have also contributed 23 protocols used in their own laboratories for the processing, handling, and induction of stem cells. Each of these protocols are annotated with the lab from where the samples originated and, if available, the individual scientists who the protocol should be attributed to. Each protocol has a DOI so future use of the protocol can be cited and attributed to the lab and individuals responsible for it’s creation.www.wicell.org). Links to WiCell are being generated to be able to order the cell lines, as well as links from WiCell back to Synapse for access to derived data.We have automatically generated curated pages (syn5762789) for each of the publicly available C4 cell lines which contains cell line and assay metadata as well as summaries and tables of links to all raw and derived files for the specific line in Synapse. Each page has a DOI for use in citation. Physical samples of cell lines have been depositied with WiCell Research Institute .Donor gender was confirmed by karyotyping (syn2679104), and lines originating from a common donor were identified using SNPs from arrays and RNA-Seq (see Methods) through an identity-by-descent analysis with PLINKP-values) as well as determine covariates that would contribute significantly to any downstream analysis . We utilized principal component analysis (PCA) and hierarchical clustering as a primary modes of assessing potential variability and major class differences among each of the analyses differentiation states  and specific donor among other signals. These analyses indicate that samples are most similar among differentiation states, suggesting this data is suitable for covariate associated comparison analyses.https://github.com/Sage-Bionetworks/synapsePythonClient, https://github.com/Sage-Bionetworks/rSynapseClient/) via the indicated Synapse accession numbers or by querying the associated metadata annotations. Examples are included for quality control and normalization of this data using IPython Notebooks and R Markdown documents for many of the provided accession numbers. Documentation about Synapse, including the clients, is available at http://docs.synapse.org/.There are multiple routes for accessing the stored data: 1) web-based file navigation, 2) programmatic access , 3) interactive gene queries and visualization, and 4) signature enrichment analysis in ToppGene. All data files can be accessed through the Synapse web interface to select individual files or directories of files containing the described materials. All files contain provenance tracking information associating each result file with its associated raw formatted file, analysis scripts used for processing, version of software used, protocols, and metadata. This same data can be accessed using the Synapse clients (https://www.synapse.org/#!Synapse:syn1773109/wiki/63531)  . We provAll assay files in this project are annotated to facilitate finding them with the information from the metadata tables. For example to find all mRNA fastq files originating from CD34+ cells , we use a Synapse query from one of the clients. For example, from the command line client (which comes with the Python client):synapse  query "select * from syn7511263 where dataType=mRNA AND fileType=fastq AND Cell_Type_of_Origin='CD34+ cells'"Similarly in Python:import synapseclientsyn = synapseclient.loginresults = syn.tableQuery("select * from syn7511263 where dataType=mRNA AND fileType=fastq ANDCell_Type_of_Origin='CD34+ cells'")or in the R client:library(synapseClient)synapseLoginresults <- synTableQuery("select * from syn7511263 wheredataType=mRNA AND fileType=fastq ANDCell_Type_of_Origin='CD34+ cells'")https://www.synapse.org/register) is required for downloading the available files.The raw and normalized data products are provided as open-access data without any usage constraints or licensing required, as per the Synapse end-user access agreement. A free Synapse account .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.