The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM. Glioblastoma multiforme (GBM) is the most common and most aggressive brain tumor in adults. It remains an incurable disease with a markedly diminished life expectancy despite multimodal therapy comprising surgical resection, chemotherapy, and radiation (RTX) ,2. RecenRTK-AXL is a transmembrane receptor with a C-terminal intracellular domain and an N-terminal extracellular domain. RTK-AXL belongs to the TAM family of kinases and has a specific ligand, which is the growth arrest-specific protein 6 (GAS6) ,14. BindIn GBM, RTK-AXL is widely expressed and activated in primary and recurrent diseases . The actFortunately, there is now a variety of AXL TKIs available whose effectiveness can be studied in detail in vitro and in vivo ,22,23. TTo evaluate the role of RTK-AXL in acquired and intrinsic therapy resistance and to provide a robust rationale for future study designs of clinical trials investigating TKIs in GBM, we aim to: (i) characterize the regulation of RTK-AXL in response to RTX and temozolomide (TMZ) treatment, (ii) investigate whether endogenous RTK-AXL expression is related to therapy response; and (iii) evaluate if the co-administration of the AXL-specific TKI R428 to standard GBM therapy, consisting of TMZ and RTX, augments treatment effects.p = not significant, First, we investigated whether the treatment with TMZ modifies the RKT-AXL expression in glioma cells. SF126 and U118MG cell lines were exposed to different TMZ concentrations, and the corresponding AXL mRNA levels were determined. Only minor changes (less than 1.5-fold) of AXL mRNA levels were observed in both cell lines following exposure to therapeutic doses of TMZ (10 µM) after 24, 48 and 72 h, and was most pronounced at 48 h (Ordinary one-way ANOVA p < 0.0001). This dose-dependent, opposed regulation of FL-AXL and CT-AXL could be interpreted as a post-translational receptor modification in response to TMZ treatment. To prove this observation, we aimed to measure the N-terminal RTK-AXL domain, also known as soluble AXL (sAXL), in the supernatant of the TMZ-exposed cells. In accordance with the increasing CT-AXL expression levels, we detected an increased activity of the tumor necrosis factor-alpha converting enzyme (TACE/ADAM17), a member of the disintegrin-metalloprotease (ADAM) family that is involved in proteolytic shedding of the RTK-AXL releasing sAXL following TMZ treatment [To simulate the clinical situation of prolonged TMZ exposure, we now exposed cell lines to increasing doses of TMZ on five consecutive days. Interestingly, FL-AXL decreased and CT-AXL increased in a dose-dependent manner E. Quantireatment . This inTo determine RTK-AXL regulation in response to RTX, the cell lines SF126 and U118MG were exposed to increasing dosages of RTX ). On mRNA level, no significant regulation of AXL mRNA was observed with the most pronounced however not significant changes 24 h after RTX A,B. CorrTo simulate a radio-resistant microenvironment, we cultivated the cells under hypoxic conditions resulting in an increased expression of hypoxia induced factor 1 , a known trigger of radio-resistance ,27,28. WTo assess RTK-AXL phosphorylation, downstream pathway activation and activation of alternative RTKs in response to TMZ and RTX, Western blot with activated Akt (pAkt) and STAT (pSTAT)  and Protein phospho-kinase array was applied. No significant changes of RTK-AXL down-stream pathway activation or phosphorylation of alternative RTKs were observed in response to the therapy .t-test SF126 vs. SF126-TR: ** p = 0.006; paired t-test U118MG vs. U118MG-TR: ** p = 0.025, p = ns, U118MG ** p = 0.0069). To study the efficacy of TKI R428 in the established TR-cells, we treated SF126-TR and U118-TR cells with R428 and TMZ and performed a CFA. Interestingly, the CFA revealed a significant reduction in the colony formation capacity in both SF126-TR and U118-TR cell lines with R428 given alone (Student’s t-test comparing SF126 with SF126 + R428: ** p = 0.0015 and U118MG with U188MG + R428: ** p = 0.0028). The combination of TMZ and R428 further increased this effect .Irrespective of RTK-AXL modifications in response to therapy and hypoxia, we aimed to determine whether simultaneous RTK-AXL inhibition with TKI R428 and TMZ/RTX could increase therapy response and if TKI R428 could aid in overcoming therapy resistance. Therefore, we established two TMZ-resistant cell lines (SF126-TR and U118MG-TR) in addition to the TZM naïve cell lines (SF126 and U118MG), to study the efficacy of the combination treatment. To create the TR cell line, SF126 and U118MG cells were treated with gradually increasing concentrations of TMZ (10–150 µM) for 10 to 12 weeks. Resistance to TMZ was based on a stable colony formation capacity in SF126-TR and U118-TR cells under TMZ treatment, compared with a significant decrease of viability in naïve SF126 and U118MG glioma cells (p < 0.0001). A similar trend was observed for the U118MG cell line; however, the results were not statistically significant (p = ns). These results indicate that the application of R428 alone in TR cells seems to be effective, whereas the combination of R428 with TZM or RTX leads to an improved therapeutic response even in TMZ resistant cell lines. This in turn supports the hypothesis that RTK-AXL inhibition might increase sensitivity towards TMZ and RTX and has the capacity to resolve mechanisms of therapy resistance.We set out to test the radio-sensitizing capacity of R428 by exposing SF126 and U118MG cell lines to RTX and R428 and quantified cell survival using CFA. The addition of R428 to RTX treatment resulted in a highly significant decrease in colony formation capacity in the SF126 cell line (p < 0.0001). With this data we show that elevated endogenous expression of the functional RTK-AXL is associated with less radiosensitivity, whereas sensitivity to TMZ remains unaffected.Lastly, we aimed to test the hypothesis whether endogenous RTK-AXL expression levels are related to treatment response. Therefore, the following modified cell lines were exposed to TMZ and RTX: SF126-DN: overexpressing the RTK-AXL lacking the intracellular domain (AXL-DN) and SF126-WT: overexpressing the functional RTK-AXL . SF126, With this study we showed that RTK-AXL undergoes post-translational degradation in response to repeated TZM exposure and under hypoxia, representative for a radio-resistant microenvironment, whereas these effects were not observed following a single fraction of RTX or single dose of TMZ. Further, we showed that high endogenous levels of FL-AXL reduced therapy response to RTX. However, therapy resistance may be overcome by combining AXL TKI R428 to standard treatment, even in TMZ-resistant GBM cell lines.Post-translational receptor modifications are considered a relevant mechanism of therapeutic resistance to TKIs ,29. AddiIn the clinical context, the question arises as to the optimal timing for the use of TKIs and appropriate combinational therapies. Our data shows that TKI R428 is acting as a therapy sensitizer to TMZ. The same has been observed in cell lines with acquired resistance to TMZ. When combining TKI R428 and RTX, we observed a significant improved therapy response of the SF126 cell line. These findings indicate that TKI R428 increases sensitivity towards TMZ and RTX and has the capacity to resolve mechanisms of therapy resistance. Since these results could not be reproduced for the combination of RTX and TKI R428 in the U118MG cell line, there might be a rationale for individual testing of patient-derived cell lines in future GBM studies to generate substantial rationales for applying AXL TKIs as single agents or to standard therapy consisting of fractionated stereotactic RTX and TMZ.Regarding patient stratification for the use of AXL TKIs in clinical trials, we addressed the role of endogenous RTK-AXL expression in GBM. Although we could not establish a correlation between high RTK-AXL expression and the TMZ treatment response, there seems to be a correlation between endogenous AXL expression and radiosensitivity, which corresponds well with previously published studies . We, aloIn summary, our data show that RTK-AXL appears to play a critical role in both intrinsic and acquired resistance to standard GBM therapy. Monitoring RTK-AXL expression as well as shedding products as indicators of therapy resistance and tumor progression could be a promising approach for individualized therapy management as it is currently being studied for malignant melanoma and hepatocellular carcinoma patients ,31.Human high-grade glioma cells U118MG were obtained from American Type Culture Collection (ATCC). Human high-grade glioma cells SF126 were obtained from the JCRB Cell Bank. Both were primary tumor cell cultures derived from surgical specimens of human GBM, WHO Grade IV. Cell authentication was carried out with LGC Standards Cell line Authentication service in June 2014 with 16 loci service of short tandem repeat profile. Further, we used a truncated form of human RTK-AXL lacking the intracellular RTK-bearing domain into SF126 cells (AXL-DN). SF126 naïve cell line served as control. Western blotting confirmed expression of the wild-type and truncated receptor in AXL-DN cell clones .2, 95% humidity in a tissue culture incubator. Growth medium encompassed Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (penicillin/streptomycin). Each experiment was conducted under the same conditions and repeated in three independent runs. Cell lines were cultivated to 80% confluence before starting an experiment. Therefore, cells were cultivated with 10–15 mL of cell culture medium in a T75 flask or with 5 mL of cell culture medium in a T25 flask. Cells were seeded at a density of 7 × 105 cells per mL medium. The cell culture medium was replaced 24 h after thawing the cells and was then replaced twice a week. Cell counts and cell viability were assessed with CASY® Cell Counter TT (OLS). For hypoxia experiments, cells were cultured under 1% O2 for 3, 6, 12, and 24 h accordingly to reports from the literature [Tumor cells were maintained as monolayer cultured in tumor growth medium at 37 °C, 5% COterature ,39.A known number of cells (250–1000) were plated into a 12-well plate and treated with either TMZ, RTX or combined treatments starting after one day in culture. After a growth phase of 12 to 14 days cells were fixed with 70% ethanol and stained with Giemsa. Surviving cell colonies were counted visually and microscopically. A colony was considered >50 cells. Survival fractions were graphed in relation to vehicle control.® Reverse Transcription Kit . Quantitative analysis was performed using QuantiTect® SYBR Green gene expression master mix  and QuantStudio™ 6 Flex Real-Time PCR System . Each reaction compound was composed of 5 µL SYBR Green master mix, 4 µL cDNA (10 ng/mL), 0.2 µL ROX lL, 0.2 µL of forward and reverse primers, respectively and 0.4 µL H2O yielding a total volume of 10 µL. Expression levels of the housekeeping gene 18S were used to normalize RTK-AXL expression levels and RQ values were graphed in relation to vehicle control. We consider a RQ significant when there is a minimum of two-fold change. The relative mRNA levels were calculated based on the cycling threshold (Ct) values. Each experiment was repeated in biological triplicates.Following isolation of total mRNA using the PureLink™ RNA Mini Kit  according to the manufacturer’s instructions, the concentration and purity of extracted mRNA was measured with Tecan Infinite Pro 200 microplate reader . Reverse transcription was carried out using QuantiTecthttps://www.ncbi.nlm.nih.gov/tools/primer-blast/ accessed on 20 January 2017) and according to Roux et al. [Primer design was done with the NCBI primer design tool . Santa Cruz: Gas6 antibody (C-20). Sigma Aldrich: mouse monoclonal β-actin antibody (clone 1A4). Immune complexes were visualized using a second HRP-conjugated anti-rabbit or anti-mouse antibody (Pierce Biotechnology). Pierce™ ECL Western Blotting Substrate  was used to develop blots and ImageJ Software was used for densitometry analysis. Reported values were first normalized to the loading control and then multiplied by a constant to reach the lowest whole integral. Each Western blot was carried out with a negative control consistent of sample diluent or beads incubated with antibody and sample diluent only. Western blot quantification was carried out using ImageJ software  according to the user guide IJ1.46r.After lysis in Radioimmunoprecipitation assay (RIPA) buffer, samples were frozen and stored in −80 °C. Protein concentrations of the samples were measured using the BCA Protein Assay as instructed by the provider . Experiments were run with 20 µgr protein. Western blots were repeated with protein samples from 2–3 independent experiments. Protein samples were boiled for 5 min with Laemmli sample buffer (Bio-Rad Laboratories) and loaded into pre-poured Tris-HCl-glycine SDS-PAGE gels . Gels run at 150 V for 1.5 h following transfer to a polyvinylidene difluoride membrane  at 400 mA constant current for 2 h. Blots were blocked with 5% BSA in 1xTBST, primary and secondary antibodies were dissolved in TBST. Following primary antibodies were used. R&D Systems: human phopho-AXL (Y779) mAb (Clone 713610), Cell Signaling: anti-AXL C89E7 rabbit mAb, abcam: anti-AXL (EPR21107) ab215205 mAb, phospho-MET (Tyr1234/1235) rabbit mAb (D26) XPTACE assay , was carried out as previously described according to manufactures instructions. Cells were lysed in lysis buffer as previously described and total protein content was measured using Pierce™ BCA Protein Assay Kit  . 50 ug pThe Proteome Profiler™ Array Human Phospho-RTK Array Kit  was used according to the manufacturer’s instructions. Protein was harvested in lysis buffer and 300 ug total protein was loaded onto specialized membranes coated with multiple human phospho-kinase antibodies. Detection was performed with GeneSnap program shooting for 20 min with 1 min running time between captures.TZM for use in cell culture was acquired commercially from Sigma Aldrich . To establish TMZ-resistant glioma cell lines, SF126 and U118MG cells were exposed to gradually increasing concentrations of TMZ (5–150 µM) in culture media for 10 to 12 weeks. TMZ was prepared freshly every day and added as 1% DMSO composite of total volume to fresh tumor growth medium. Vehicle control cells were fed daily with fresh tumor growth medium containing 1% DMSO. For two weeks cells were exposed to low-dose concentrations of 5 µM TMZ. Then, concentrations were increased prior to every other passage when cells had reached 85% confluence. Cells were passaged in half to ensure sufficient survival. Later during high-dose treatment (>80 µM) cells were given multiple passage cycles before rising in treatment concentration. The generated TMZ-resistant cell lines were named SF126-TR and U118MG-TR. Cells were assessed regularly for sufficient viability using colorimetric microtiter (MTT) assay and microscopy .® Cell Counter TT (OLS) in 12-well plates for CFA or 25 cell culture flasks for Western Blot and PCR experiments and received different RTX doses  after 24 h in culture. Applied RTX doses were determined according to other studies investigating RTX effects in glioma cells [Human high-grade glioma cells SF126 and U118 were plated at a defined density measured with the CASYma cells . Cell dama cells .RTX dose was applied at room temperature using the Xylon Maxi shot  at 200 KV, 10 mA at a rate of 1 Gy/1:08 min. Irradiated cells were further used for Western blot analysis or real time PCR as described above 24 or 48 h after RTX. Radiosensitivity was assessed using the CFA.R428  was purchased from Selleckchem  and was dissolved in dimethyl sulfoxide . Stock solutions of 1 mM were prepared according to the manufacturer’s instructions, and R428 was diluted in Dulbecco’s modified Eagle medium (DMEM) to final concentrations of 0.5 µM, 1 µM, and 2 µM. Human glioma cells were treated with a single dose of R428. t-Test and one-way ANOVA combined with Bonferroni’s multiple comparison test. Significance level was set at alpha = 0.05 . The level of significance was set at p < 0.05. Data were expressed as mean +/− standard deviation. Quantitative and qualitative statistical analyses were performed using GraphPad Prism 5 .Each group consisted of at least triplicates. Statistical analysis was performed using GraphPad Prism, Version 7.0c. Statistical significance was measured using Student’s


Arabidopsis thaliana. Analyses of multiple homozygous lines depleted of phytochromes in stomatal guard cells indicated elongated hypocotyls specifically in red and far-red growth conditions. Furthermore, rosette leaves of adult plants with guard cell-specific phytochrome deficiencies showed enhanced serration compared to the wild-type Col-0 parent. Thus, we demonstrate that guard cell-localized phytochromes impact the inhibition of hypocotyl elongation, as well as leaf margin morphology of adult rosette leaves in A. thaliana.Using a previously established transgenic approach to inactivate phytochrome chromophore synthesis in specific organs or tissues, we used a guard cell-specific promoter to induce phytochrome deficiencies in guard cells of  Light actively inhibits elongation in the hypocotyl, but promotes the growth and development of cotyledons, leaves, and roots. Distinct responses in specific tissues are maintained to some extent through the differential accumulation of photoreceptors such as phytochromes in distinct tissues and at different times during development . Specific phytochromes have been shown to impact stomatal development and function, with phyB promoting the stomatal index, or the percentage of stomata cells out of the total number of epidermal cells, and regulation of stomatal opening in red light . The photoreceptor phyA stimulates production of stomata in far-red light . Notably, phytochromes are localized in guard cells themselves , and thus stomatal responses can potentially be controlled by photoreceptors locally or through intercellular communication.Stomata are specialized epidermal cells that occur in the epidermis for all above-ground tissues and serve to facilitate gas exchange and water uptake central to photosynthesis. Stomata respond to environmental signals, including light  expression to explore phytochrome-dependent roles in guard cells . Expression of PHYB in guard cells demonstrated that phyB accumulation in these cells was sufficient to rescue a defect in photoregulation of the stomatal index in a phyB mutant . These plant lines with guard cell-specific PHYB expression also exhibited larger leaves, potentially indicating more extensive growth-related impacts of guard cell-specific phytochrome function. Prior investigations also correlated stomatal regulation with control of additional whole plant growth phenotypes, including correlating the regulation of stomatal opening with vegetative development in terms of the hypocotyl length of seedlings , and the rosette and flowering time at the adult stage .Tissue-specific expression of et al., 1997; Montgomery et al., 1999; Montgomery et al., 2001; Warnasooriya and Montgomery, 2009; Costigan et al., 2011). We used the promoter pGC1 (At1g22690), which drives high-level, guard-cell specific gene expression and has low expression activity in mesophyll cells , to generate plant lines with guard cell-localized, plastid-targeted BVR (pBVR) accumulation to induce guard cell-specific phytochrome depletion. We used these lines to examine the roles of guard cell-localized phytochromes in distinct aspects of photomorphogenesis.To investigate the function of phytochromes in guard cells, we used a previously verified transgenic approach for expressing a phytochrome chromophore-inactivating enzyme, biliverdin reductase  to regulate accumulation of photoactive phytochromes in planta . We observed significant enrichment of BVR expression in GCEEP compared to MC in several GC::pBVR lines, confirming guard-cell enriched BVR expression driven by the GC promoter. To confirm the reliability of our MC and GCEEP fractions, we tested RNA fractions for expression of an MC-enriched marker gene, i.e., CALMODULIN-BINDING PROTEIN  , and guard-cell marker genes, i.e., POTASSIUM (K+) CHANNEL IN ARABIDOPSIS THALIANA 1   and MYB DOMAIN PROTEIN 60  . As expected, we detected preferential expression of CBP in the MC RNA enrichment and of KAT1 and MYB60 in the GCEEP RNA enrichment . Additionally, we assessed protein extracts from GCEEP for BVR enzymatic activity using a previously described specific activity assay . Whereas no BVR activity was detected for GCEEP protein extracts from WT and a CAB3::pBVR line, we detected BVR activity in the range of 0.2 – 2.8 (I.U. mg−1) × 103 for GCEEP extracts from GC::pBVR lines.We successfully isolated four lines with guard-cell enriched expression of BVR, i.e., GC::pBVR1 to GC::pBVR4 as verified by qRT-PCR analyses with RNA extracted from mesophyll cell (MC) and guard cell enriched epidermal peel (GCEEP) fractions and compared to MC and GCEEP fractions from the CAB3::pBVR representative line with MC-localized et al., 2011). The disruption in light-dependent inhibition of hypocotyl elongation only under R and FR light for GC::pBVR lines is notable as these are wavelengths specifically associated with phytochrome-regulated functions. Although not previously associated specifically with light-dependent regulation, prior analyses of plants with altered ABA-regulated stomatal function showed a correlation between higher rates of stomatal closure and elongated hypocotyls .We assessed light-dependent growth of GC::pBVR seedlings compared to Col-0 WT seedlings under constant blue (Bc), red (Rc), far-red (FRc) and white (Wc) light. We observed significantly elongated GC::pBVR seedlings under both FRc and Rc illumination . GC::pBVR seedlings were not significantly different relative to WT seedlings in the dark or under either Bc or Wc light , which is distinct from phenotypes observed due to depletion of phytochromes in mesophyll cells as CAB3::pBVR lines were previously reported to have elongated hypocotyls in Wc, FRc, Rc, and Bc . In Arabidopsis, leaf heteroblasty can be indicated by leaf margin serration. As the major disruption at the adult leaf stage for GC::pBVR lines was an alteration in leaf serration or leaf margin morphology defects, these plants may exhibit alterations in heteroblasty correlated with guard-cell phytochrome deficiency. Relatedly, prior analyses with tissue-specific expression of phyB also indicated a correlation between a role for guard cell-localized phyB and leaf phenotypes. The leaves of phyB mutants are significantly different from WT with less leaf area and elongated petioles . When a phyB mutant was specifically complemented with a guard cell-localized PHYB construct, i.e., SPCH::PHYB, the leaf phenotype was largely complemented, in addition to the stomatal index phenotype . Together, these associations of the regulation of leaf phenotypes with stomatal phytochrome pools indicate key roles of GC-localized phytochromes in regulating whole leaf phenotypes.For adult plants, GC::pBVR plants exhibited altered leaf shape  under long-day conditions of W light at 100 µmol mPlasmid constructionBVR fragment for plastid-targeted BVR protein accumulation from the previously described CAB3::pBVR construct pBIB/CAB3-TPBVR  and guard cell-specific GC promoter . Specifically, the guard-cell specific, plastid-targeted BVR construct (GC::pBVR) was made by isolating the transit peptide-BVR (pBVR) fragment from pBIB/CAB3-TPBVR using XbaI and SacI and subcloning into XbaI and SacI digested pORE_O3_GC plasmid, which contains the GC1 promoter [pGC1 (At1g22690)], by ligation to create the pORE_O3_GC/TPBVR plasmid.We constructed plant transformation vector pORE_O3_GC/TPBVR using a TP-Plant transformation and growth conditionsArabidopsis thaliana (Columbia or Col-0 ecotype) plants were used as wild-type (WT) and for transformation of all constructs. We used both pBIB/CAB3-TPBVR and pORE_O3_GC/TPBVR constructs in Agrobacterium tumefaciens strain GV3101. We isolated multiple single insertion homozygous lines that were screened for BVR expression, including enrichment in the mesophyll or guard cells for the CAB3::pBVR4  and GC::pBVR lines, respectively.For hypocotyl growth analyses, surface sterilized seeds were plated and grown on MS medium containing 1% (w/v) sucrose and 0.7% (w/v) Phytoblend agar  at 22°C for 7 d under the indicated light conditions. Light sources used included blue , far-red , red , and white (W) as previously described . Light intensities were measured using a LI-COR LI-250A Light Meter with inline LI-COR quantum sensor for R, B and W light, and using a StellarNet EPP2000 spectroradiometer (Apogee Instruments) for FR light.Phenotyping-2 s-1 and 22°C/20°C day/night temperature cycles.We determined light-dependent hypocotyl elongation of seedlings under specified growth conditions by scanning images of seedlings and quantifying hypocotyl length using ImageJ software as previously described . For phenotypic observation of rosette leaf morphology, plants were grown in a long-day growth chamber with 16 h/8 h day/night light cycle at a white light intensity of ~100 µmol mGene expression analysiset al., 2007) or guard cell-enriched epidermal peels from adult plants grown in a short-day growth chamber with 8 h/16 h day/night light cycles at a white light intensity of 100 µmol m-2 s-1 and 22°C/20°C day/night temperature cycle, according to the protocol of . Quantitative RT-PCR (qRT-PCR) was performed essentially as described . Expression of BVR and tissue-specific marker genes was assessed, including mesophyll cell-enriched marker CBP (At4g33050)and guard cell marker genes KAT1 (At5g46240) and MYB60 (At1g08810). The control genes used for qRT-PCR analyses were PP2A (At1g13320) and ACT2 (At3g18780). Primers for each gene are listed in Table 1.RNA samples were extracted using the RNeasy® Plant Minikit  as previously described  from whole plants, mesophyll cells from ~15 g of 5 to 6 week-old plant leaves (Yoo Table 1. Primers for quantitative reverse transcriptase PCR (qRT-PCR)


Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases, including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.The past decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed the Integrated Collection of Stem Cell Bank data (ICSCB) ( •Searches >16,000 stem cell lines in Europe, Japan, and US major databases•Data formats standardized by minimum items in MIACARM guidelines•Searches specific stem cell lines according to disease, donor, tissue, etc.•User-friendly website accesses >6,000 diseased stem cell lines from 36 countries This new and largest stem cell data portal can retrieve >16,000 cell lines from four major stem cell data resources in Europe, Japan, and the United States, is automatically updated to provide the latest cell line information, and, through its integrative search engine, can collect cell line information from specific donors with rare diseases worldwide. Since the first report of human induced pluripotent stem cells (iPSCs) , there hhttp://icscb.stemcellinformatics.org/ or, as a slim version by removing cell line redundancy as much as possible, through the SHOGoiN  homepage at http://shogoin.stemcellinformatics.org/.In this paper, as our next step toward the unification and utilization of the stem cell line data in the world, we report our new database portal, Integrated Collection of Stem Cell Bank data (ICSCB), which was designed using MIACARM guideline items and formats to serve as an entrance “port” to individual data repositories. The main objectives of ICSCB are (1) to establish an integrated stem cell database portal that can cover the majority of stem cell resources in the world and (2) to offer users minimum but efficient access to information on stem cell lines based on MIACARM guidelines. Currently, ICSCB provides data on more than 15,000 stem cell lines registered in four major stem cell line databases: hPSCreg , SKIP K, RIKEN BICSCB was designed for researchers searching for available cell lines to conduct various studies, such as regenerative medicine and disease analysis. Covering as many diverse cell lines as possible was the first priority when deciding which resources to include in ICSCB. Sharing cell line information between different stem cell banks and registries has been problematic due to different cell naming methods, different policies on cell assessment in different registries, unclear data sources, and so on. ICSCB is a collection of cell lines from four major and reliable cell line data resources based in Europe, Japan, and the United States. ICSCB updating is regularly performed for new SKIP and eagle-i stem cell line data as well as automatically performed for hPSCreg and RIKEN BRC data in a synchronized manner. Users can retrieve all related stem cell line information by using a free text search. Detailed information on a specific cell line can be accessed by clicking on the stem cell ID, which is linked to the information page in the original resources . The resICSCB covers more data than any other stem cell line repository available. The integration of all major data resources allows us to check the current state of stem cell research in the world . Althoughttps://stemcellinformatics.org/). SHOGoiN is a repository for accumulating and integrating diverse human cell information to support a wide range of research using cell-related data. The database consists of several modules that store cell lineage maps, transcriptomes, methylomes, cell conversions, cell type markers, and cell images with morphology data curated from public as well as contracted resources based on sophisticated cell taxonomy. Collaboration between ICSCB and SHOGoiN makes it possible for users to directly use free text searches for stem cell line data on the SHOGoiN homepage. The ICSCB easy search module in SHOGoiN supports a simplified ICSCB search with keywords, and the advanced search is designed to redirect users to the ICSCB homepage with full functions. Results from the SHOGoiN homepage have the same structure as in the ICSCB homepage.ICSCB also has a quick and easy search module on the SHOGoiN homepage  SKIP , (2) hPSCreg , (3) RIKEN BRC , and (4) eagle-i  . These data resources were selected because they had the highest number of registered cell lines and a large diversity, which would provide a good regional balance of cell sources to reduce redundancies in cell line entries. RIKEN BRC basically collected cell lines from Japanese institutions, SKIP contained data mostly from other Japanese and Asian institutions, hPSCreg collected data mainly from European institutions, and eagle-i collected data mostly from the United States. Details of the data sources are listed in Since our previous research on the listed stem cell banks 4 years ago , the numTo provide fast and easy access to the latest and accurate cell line information, we built an automatic updating system that adds newly released cell lines to ICSCB as soon as they become available in any of the four data resources. Data from eagle-i and SKIP are directly collected and stored in the MySQL database with the terms required for the MIACARM modules. Data from hPSCreg and RIKEN BRC are collected on the fly per request using a web application programming interface (API) provided by the respective sites. RIKEN BRC also uses SPARQL language for data retrieval requests .SOX2) such that the results page will display relevant entries by showing the indicated keyword in the extra field below (To simplify the search process, ICSCB provides an easy-to-use and mobile-friendly web application. The goal of the application is to help users find the desired stem cell lines as quickly as possible. The interface of the search engine is designed with the 16 MIACARM terms , except ld below B. Furthein vitro, morphology data, marker gene expression/surface antigen expression data, karyotyping assay results, copy number variation, residual exogene detection results, genome profiling, transcriptome profiling, and epigenome profiling data are accessible from ICSCB.ICSCB also provides a quality control panel based on MIACARM, thereby supporting customized searches according to quality control results. At present, assays for teratoma formation, differentiation ability https://github.com/YingChen-bio/ICSCB.The original data and R source code for creating figures and supplementary figures and tables are available at Y.C. and Y.P. drafted the manuscript. S.M., T.M., H.O., J.D., S.S., A.K., H.M., Y.N., M.S., J.S., and W.F. provided and facilitated the stem cell data. W.F. and K.S. conceptualized the research. A.K., G.S., and W.F. led the project.H.O. is a founding scientist of SanBio Co., Ltd., and K Pharma, Inc.


Cancer cell lines are a defined population of cells, originally sourced from tumour tissue, that can be maintained in culture for an extended period of time. They are a critical laboratory-based model, and are frequently used to unravel mechanisms of cancer cell biology. In all cells, gene activity is in part regulated by DNA methylation, the epigenetic process by which methyl groups are added to DNA. In this study, we demonstrate that at a global level, DNA methylation profiles are globally well conserved, but we identify specific sites that are consistently more methylated in tumour-derived cell lines compared to the original tumour tissue. The genes associated with these common differentially methylated regions are involved in important cellular processes and are strongly enriched for epigenetic mechanisms associated with suppression of gene activity. This study provides a valuable resource for identifying false positives due to cell culture and for better interpretation of future cancer epigenetics studies.DNA methylation is a heritable epigenetic mark that is fundamental to mammalian development. Aberrant DNA methylation is an epigenetic hallmark of cancer cells. Cell lines are a critical in vitro model and very widely used to unravel mechanisms of cancer cell biology. However, limited data are available to assess whether DNA methylation patterns in tissues are retained when cell lines are established. Here, we provide the first genome-scale sequencing-based methylation map of metastatic melanoma tumour tissues and their derivative cell lines. We show that DNA methylation profiles are globally conserved in vitro compared to the tumour tissue of origin. However, we identify sites that are consistently hypermethylated in cell lines compared to their tumour tissue of origin. The genes associated with these common differentially methylated regions are involved in cell metabolism, cell cycle and apoptosis and are also strongly enriched for the H3K27me3 histone mark and PRC2 complex-related genes. Our data indicate that although global methylation patterns are similar between tissues and cell lines, there are site-specific epigenomic differences that could potentially impact gene expression. Our work provides a valuable resource for identifying false positives due to cell culture and for better interpretation of cancer epigenetics studies in the future. DNA methylation is a stable and somatically heritable epigenetic modification. In mammals, DNA methylation involves attachment of a methyl group to a cytosine, usually in the context of a CpG dinucleotide. Methylation patterns are established during early embryogenesis and play a key role in cellular differentiation, lineage commitment and maintenance of cell identity ,2. In soAlthough cancer cell lines have been an indispensable and a very widely used model for cancer biology studies for decades, they often do not fully represent the epigenetic state that exists in vivo ,17,18. IHere, we provide the first genome-scale methylation profiles of metastatic melanoma tumour tissues and the corresponding cell lines derived from these tissues using reduced representation bisulfite sequencing (RRBS) . RRBS isRRBS was used to establish genome-wide DNA methylation maps of melanoma tissues (53T and 60T) and their derived cell lines , producing a total of 101.4 million 100 bp sequenced reads that were then mapped to the GRCh37 reference human genome . We analr = 0.96, r = 0.87, As expected from previous analyses of human cell lines and somatic tissues , the metWe observed that both the cell lines were globally hypermethylated as compared to their corresponding tumour tissue. In both cases, we identified 10% higher median methylation in cell lines compared to tumour tissues  in the comparison between 53T and 53C and 13,089 DMFs in the comparison between 60T and 60C . All DMFConsistent with the global methylation patterns, we found that a much higher proportion of DMFs were hypermethylated in the melanoma cell lines. Compared to 53T and 60T, 88% and 83% of the DMFs were significantly hypermethylated in the cell lines, respectively . The metp-value < 0.01, SUZ12 and EZH2 . However, this is consistent with known inter-tumour heterogeneity of methylation levels. A recent whole genome bisulfite sequencing study which profiled nine cell lines from different tumours reported global methylation levels in those lines that ranged from 41.8% to 65.4% .In this study, we identified 783 regions that showed common differential methylation between both the analysed pairs of cell lines compared to their tumour tissue of origin and 94.1% of these regions were hypermethylated in the cell lines. In a previous study, RRBS analysis was performed between embryonic stem cells (ES) and ES cells differentiated in vitro into neural precursor cells (NPC). The global methylome was significantly correlated between the ES cells and in vitro derived NPCs (rho = 0.81). However, a much higher number of CpGs (~8%), that were unmethylated in ES cells, became methylated in NPCs, and a relatively smaller proportion of CpGs (2%) that were methylated in ES cells, became unmethylated in NPCs . FurtherPrevious studies have identified common hypermethylated loci in multiple cell lines, when compared to tumour tissue . Here, wDNA methylation establishes and stabilises cellular phenotypes by maintaining gene expression states, which means that specific DNA methylation patterns are characteristic of particular cell types, lineages and phenotypes . AlthougWe discovered that the genes associated with the common site-specific methylation differences between cell lines and their corresponding tumour tissues are very strongly enriched for the repressive H3K27me3 mark. Polycomb Group (PcG) proteins including Polycomb Repressive Complexes, PRC1 and PRC2 are widely recognized to mediate gene silencing of developmental genes and also play a role in repressing genes in cancer ,58. One It was reported that for a set of genes highly enriched with H3K27me3, CGI promoters were aberrantly hypermethylated in cancer cells but in not in normal cells . AlthougWe have analysed two pairs of tissues and their corresponding derivative cell lines, and it could be argued that this is a low sample number. However, such materials  are very rare, and to our knowledge, this study provides an RRBS map of tissues and matched derivative cell lines for the first time, for any tumour. Importantly, in both of our pairs independently, we observed consistent patterns of methylation, and overall conservation of global methylation, consistent with site-specific hypermethylation in the cell lines compared to the tissues. These results further indicate a common pattern of methylation in cell lines, as compared to tumour tissues in this study. A potential limitation is the absence of gene expression data for these samples. However, our hypothesis was that gene expression is a consequential event to genetic and epigenetic alterations, and we primarily wanted to investigate the nature of conservation and changes in DNA methylation, due to the establishment of cells in in vitro culture. Future studies with gene expression profiles of matched tissues and derivative cell lines will provide additional insight into the changes in expression program, which are due to establishment of cells in in vitro culture.The New Zealand melanoma (NZM) tissue samples and their derivative cell lines used in this study were from surgical samples of two patients diagnosed with metastatic melanoma, coded as NZM53 and NZM60. For this paper, the NZM53 and NZM60 cell lines are designated 53C and 60C, respectively, to distinguish them from their tumour tissues of origin, 53T and 60T, respectively. The original tumour samples were obtained with written consent from each patient under the guidelines and specific approval of the Auckland Area Health Board Ethics Committee, New Zealand.According to comprehensive molecular profiling of NZM cell lines , SequenoDNA was extracted from freshly cut sections of formalin-fixed paraffin-embedded (FFPE) melanoma tissue with the QIAamp DNA FFPE tissue kit, using xylene to dissolve the paraffin . The twoMelanoma cells were isolated from pathologically-confirmed metastatic malignant melanomas and long-term cell cultures were established as previously described . For thiWe used reduced representation bisulfite sequencing (RRBS) to map genome-wide DNA methylation as we have described previously ,69,70,71As previously described, the sequenced RRBS reads were quality checked using FastQC (RRID:SCR_014583) and processed using bioinformatics tools developed in house ,30. The identgeneloc program of the DMAP tool [https://genecards.weizmann.ac.il/geneloc/index.shtml, accessed on 7 April 2017), super-enhancers , CTCF binding sites  and specific repeat classes .We used our in-house Differential Methylation Analysis Package (DMAP) to identify RRBS fragments that were differentially methylated between the tissues and their derived cell lines . BrieflyMAP tool . We usedWe used Metascape (RRID:SCR_016620)  and EnriIn conclusion, our sequencing-based genome-scale analysis demonstrates a global conservation of DNA methylation patterns following the establishment of in vitro cell cultures, which had been derived from matching human metastatic melanoma tissues, and therefore provides supporting evidence in favour of using cell lines as in vitro models for methylation studies in cancer epigenetic research and drug discovery. Our results also provide an impetus for conducting similar studies in other cancer types to assess methylation patterns. We also demonstrate region-specific epigenomic differences between the cell lines and the corresponding matching tumour tissues, and reveal novel genomic features of these regions, thus providing a valuable resource for cancer methylome studies to reduce false-positive candidates from the epigenetic effects of cell culture. This resource will allow better interpretation of cancer epigenetics studies in future.


Graft-versus-host disease (GvHD) of the skin is a severe allo-immune reaction and complication following allogeneic stem cell transplantation. Over the past years, intensive pre-clinical research has led to an improved understanding of the pathophysiology of acute and to a lesser extend chronic GvHD. This has translated into the approval of several new agents for the treatment of both forms of GvHD. This review summarizes the most recent advances in underlying pathomechanisms, clinical trials and newly approved agents for GvHD, with a special focus on skin involvement. Graft-versus-host disease (GvHD) remains a frequent complication following allogeneic stem cell transplantation (HSCT). Despite better pretransplant conditioning, prophylaxes and graft manipulation, treatment refractory GvHD accounts for 5-30% . Skin isWhile up to 50% of patients after allogeneic HSCT are affected by any type of GvHD , cutaneoPathomechanisms driving GvHD need to be discussed in light of host and donor characteristics. Host predisposing factors refer to the inflammatory milieu at the time of HSCT. A certain degree of skin toxicity, i.e. inflammation or damage, will inevitably follow treatment with chemotherapeutic agents. Upon infusion of donor stem cells, the presence of chronic viral infection and an altered host microbiome may further contribute to a favorable host environment facilitating GvHD.via pathogen-associated molecular patterns (PAMP) and danger-associated molecular patterns  released from injured cells  classification system, acute GvHD may occur as classic aGvHD within the first 100 days post HSCT, or persistent/recurrent or late-onset aGvHD, reflecting clinical signs of acute GvHD beyond 100 days following HSCT. The latter is typically observed after reduction of immunosuppressive therapy. Chronic GvHD can be divided into the classic form, without signs of aGvHD and the overlap syndrome, combining features of chronic and acute GvHD .In aGvHD the extent or severity of organ involvement falls into 4 stages, and categories are defined for skin, gut and liver , 11. ForWhile highly desired, there is currently no available tool to predict the occurrence of GvHD safely. A standard GvHD prophylaxis for patients following transplantation consists of a combination of immunosuppressive agents . For manThe choice of treatment greatly depends on the grade and localization of GvHD. For locally restricted forms of grade I acute skin GvHD, the initial treatment approach will consist of a topical steroid . Low-potA well-established therapeutic option is extracorporeal photopheresis (ECP) . ParticuAntithymocyte globulin (ATG) is also commonly used in patients with steroid-refractory aGvHD. While some older studies have presented data that may indicate a less than expected effect and a higher rate of infections, more recent studies have shown better outcomes [summarized and discussed in ]. RecentThe lack of an internationally accepted primary endpoint in clinical trials of cGvHD makes the comparability of previously published trials difficult and the chosen primary endpoints may not automatically translate into a clinical benefit. While this issue has been increasingly addressed in the past years and we are now starting to obtain data from RCTs and discussions on defining a gold standard for the endpoint of these trials . FurtherFirst-line therapy in the case of a mild cGvHD are topical steroids, mostly higher potency steroids . Cutaneous atrophy or local infections may occur during long-term use of topical steroids; topical calcineurin inhibitors (tacrolimus and pimecrolimus) may spare steroids in some of these patients .Systemic steroids alone or in combination with calcineurin inhibitors are required for severe cGvHD . HoweverIn a more recent randomized control trial in cGvHD, ECP was added to the standard of care in a first line setting of patients newly diagnosed with moderate or severe cGvHD . At 28 wIn an extensive review of 27 studies, where ECP was used in steroid-resistant or dependent cGvHD, the mean response rate for cutaneous cGvHD was 74% . A compaNotably, a recent systematic review of adverse events in second- and third-line treatments for acute and chronic GvHD revealed a lower incidence of infectious adverse events in acute GvHD and lower number of grade 3-5 adverse events in cGvHD in patients on ECP compared to any pharmaceutical management highlighting the safety aspect of this form of treatment .Another therapeutic approach is the use of Rituximab. The anti-CD20 monoclonal antibody is widely used especially in sclerosing cGvHD of the skin, fasciitis, musculoskeletal cGvHD. Clinical responses up to 70% have been described . In addiIn the past years a number of novel agents have been tested in advanced clinical trials in acute or chronic GvHD and some of these drugs are now available for clinical use. While none of these trials was specifically designed only to assess the effect on skin GvHD, a large percentage of subjects included in these trials were affected by skin GvHD Table 1.Ruxolitinib, an inhibitor of the Janus kinase (JAK) 1/2, was approved for the treatment of steroid-refractory acute GvHD in 2019. JAK signaling plays an important role in regulating the underlying immune cells relevant for GvHD such as dendritic cells, macrophages as well as B- and T cells. Most importantly, there is preclinical evidence to suggest that inhibition of the JAK pathway does not negatively impact the GvL effect [as summarized in ].Approval followed a successful open-label, single arm, phase 2 trial (NCT02953678) . Of the A number of IL-2R antibodies, namely daclizumab, basiliximab and inolimomab, have been tested in clinical trials for the treatment of aGvHD. These agents generally showed some level of clinical activity in treating aGvHD, especially in cases where skin or gut was involved, , 47. HowThe TNFa antibodies infliximab and etanercept have both been investigated for the treatment of aGvHD. Studies with infliximab have delivered mixed results with a modest effect but a generally high rate of infectious complications, , 51. TheCombining TNFa with IL-2R blocking may be attractive. A prospective trial assessing patients treated with basiliximab in combination with etanercept had an ORR at day 28 of 91% and a two-year OS rate of 55% . in patients with steroid refractory GvHD , 56. Sev. In a pr. Most re. ORR at In addition to the well-established first-line treatment with prednisolone and CNIs, several new agents have recently been approved for the treatment of cGvHD.In 2017 ibrutinib was approved as a second-line treatment for steroid-resistant cGvHD. Approval came following a successful phase I/II trial (NCT02195869). Forty-two patients with active cGvHD were enrolled who had previously failed 1 to 3 prior treatments. After a median follow-up of 13.9 months the best OR was 67% . NotablyIn addition to its positive clinical activity in aGvHD, there are data from a retrospective survey, which indicates that ruxolitinib leads to a high response and 6-month survival rates in heavily pretreated patients afflicted with chronic GvHD . Based oBelumosudil is a serine/threonine kinase inhibitor blocking the activity of Rho-associated coiled-coil kinase 2 (ROCK2). The efficacy of belumosudil was evaluated in a phase 2 randomized multicentre trial in cGvHD-patients who had receive 2-5 prior lines of therapy (NCT03640481). Primary endpoint of this study of the best ORR, which was 74% and 77% for belumosudil at 200 mg daily and at 200 mg twice daily, respectively . Of noteAn innovative cellular approach in the treatment of GvHD is the adoptive transfer of regulatory T cells (Tregs). Tregs are a subset of CD4 T cells with an immunosuppressive and immunoregulatory function , 66. TheMild acute GvHD of the skin can usually be controlled with standardized treatment protocols. Severe acute skin GvHD requires more aggressive conventional treatments, but newer approaches including cellular therapies are now available. Chronic forms of skin GvHD are the transplant physician’s enemies. Functional impairment and decreased quality of life need to be avoided strenuously. Marked improvement may be obtained with extracorporal photopheresis. Drugs targeting either B cells or the JAK-STAT pathway have opened new treatment perspectives. Regulatory T cells may reverse cGvHD of the skin, but data are still preliminary. It is encouraging that patients who have overcome cGVHD, experience a health status and quality of life similar to those HSCT patients without a history of cGvHD .All authors have contributed equally in designing the review article, writing and revising. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compounds were more toxic to melanoma cells in vitro than commonly used anti-melanoma agent dacarbazine as measured by TUNEL assay. They induced both caspase-independent apoptosis evident by colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured by colocalization of TUNEL with caspase-activated DNase (CAD). In addition, compounds 3 and 5 strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). Dacarbazine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds 3, 4 and 5.The discovery of chimeric anti-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells  Malignant melanoma (MM), also known as cutaneous melanoma, is a cancer that originates from melanocytes . MelanomThiazole-derived compounds are well-known among drugs, natural products and pharmacologically active synthetic molecules . Severalhttps://dtp.cancer.gov/). These compounds were tested for cytotoxicity at a single dose of 10 µM. Several compounds showed potent activity against several cancer cell lines, and some of them were particularly potent against melanoma cell lines. Eight of these chimeric compounds (50), total growth inhibition (TGI) and 50% lethal concentration (LC50) values. Based on these data, we found a very promising structure–activity relationship of these molecules against melanoma cell lines. Overall, amino-thiazole derivatives (1–7) [8) [50 values as low as 3.22 µM. Phenyl ethyl derivative (1) inhibited the growth of all the cell lines except SK-MEL-2. Phenyl substituted derivative (2) showed good activity against LOX IMVI and MDA-MB-435 cell lines with GI50 values of 3.22 and 3.49 µM, respectively. This molecule (2) showed moderate growth inhibition against M14 and UACC-62 cell lines with GI50 values less than 10 µM. Monosubstituted phenyl derivatives 3, 4 and 5 showed very significant activities against all the tested cell lines with GI50 values as low as 1.32 µM for SK-MEL-28. 2,4-Difluoro substituted phenyl derivative (6) is found to be a good growth inhibitor of LOX IMVI and SK-MEL-5 cell lines with GI50 values of 1.71 and 1.88 µM, respectively, while it failed to show any significant activity against other cell lines. 2,4-Dimethyl substituted derivative (7) is a potent growth inhibitor of all the tested cell lines of the melanoma panel. Among all the reported thiobenzamide-derived thiazoles [8) showed significant activity against the melanoma panel.In our effort to develop new methodologies to synthesize bioactive compounds –14, we hompounds  were selivatives , 1–7 [151–7) [8) . Fluoro hiazoles , only 3-2) showed weak TGI values (greater than 48 µM) but compound (6) did not show any useful TGI values against the cell lines . N-Phenylethyl derivative (1) had the TGI values 3.17 and 6.82 µM against LOX IMVI and MALME-3M cell lines, respectively, but did not show any significant TGI against any other cell lines (3) substituted phenyl derivative (3) showed good TGI value against LOX IMVI cell line and moderate values against M14, SK-MEL-28 and SK-MEL-5 cell lines. A similar pattern was observed for other compounds .Compound  showed potent LC50 values against LOX IMVI and MALME-3M cell lines. Based on the GI50, TGI and LC50 values, compounds 3 and 5 are the most promising agents in the series. Although our compounds were most potent against LOX IMVI cell line, these cells are semi-adherent, which limited our ability to fix these cells. Hence, for the proof of concept studies below, we used adherent cell lines SK-MEL-5 and SK-MEL-28.These potent compounds also showed significant LCntration . CompounN-Aryl compounds with a small hydrophobic substituent such as fluoro, methyl and trifluoromethoxy groups showed significant activities. 3-Hydroxy substituted phenyl group is an exception.Based on the anti-melanoma potency, we can deduce a structure activity relationship (SAR) of the compounds. Aliphatic substituted compounds did not show any significant anti-melanoma activity. 2) inhibited the growth of HFF-1 cells with an IC50 value of 21 µM, which was much higher than the GI50 values against some of the melanoma cell lines. Trifluoromethoxy-substituted compound (3) did not show any notable toxicity against the tested healthy cell line, which is very important for the further drug development of this compound. 3-Fluorophenyl derivative (5) was somewhat toxic to HFF1 cells. Compound 7 showed good tolerance to healthy cell lines with the IC50 value of 40 µM. Like compound 3, the positive control, dacarbazine, also did not show any notable toxicity against HFF1 cells. IC50 values of compounds 1 and 8 were not determined against the HFF-1 cell line for their low solubility in the growth media.To assess the potential use of lead compounds as anti-melanoma agents, we tested their toxicity for the normal human foreskin fibroblast (HFF-1) cell line taking into consideration that MM is usually surrounded by the cells of this kind. The effects of some of the potent compounds on this cell line were very encouraging . Phenyl-3 caused 23% apoptosis at 6 µM and 54% apoptosis at 12 µM (5) was a very potent apoptosis-inducing agent that caused 100% cell death at 12 µM concentration. Dacarbazine, the positive control, caused 22% apoptotic cell death at 200 µM concentration. This very high concentration effected 22% apoptosis, which shows the high potency of our novel compounds against melanoma cell line.After determining the selective toxicity of potent molecules, we determined their mode of action against the melanoma cell line. Apoptosis or programmed cell death is a physiological process to remove unwanted or damaged mammalian cells. We detected apoptosis mode of cell death by our potent compounds using flow cytometry-based Annexin V assay. Compound at 12 µM . 3-Fluor3, 4 and 5) were cytotoxic to melanoma cells , and the cytotoxicity seemed to be dose-dependent. Surprisingly, the cytotoxicity of dacarbazine was negligent, and at lower concentration, it even seemed to be cytoprotective. In all three compounds as well as in dacarbazine, the cytotoxicity at 10 µM was higher than at 5 µM concentration. Interestingly, the cytotoxicity of the two test compounds was higher than dacarbazine (our positive control). It did not reach statistical significance for compound 3 at 10 µM versus dacarbazine at 10 µM, but it was significant in two other compounds (4 and 5) versus dacarbazine at 10 µM. Based on these data, and taking them together with the selectivity towards melanoma versus non-melanoma cells, we concluded that our new compounds have promise as new anti-melanoma drugs. Immunostaining for major marker of oxidative injury, heme oxygenase-1 (HO1) was done to determine whether the test compounds induce oxidative injury. The measurement of mean intensity of colour, which illustrates the level of protein expression, showed it was induced by the exposure with the two out of three compounds . Contrary to HO1, no induction of CAD or EndoG was observed . Apparently, none of these compounds induced cell death endonucleases. Activation of endonucleases and their translocation to the nucleus is actually the last step when cell death becomes irreversible. It is often preceded by an oxidative cell injury that activates or aggravates the cell death mechanism. To measure whether our compounds induce oxidative injury, we chose to measure HO1 which is a well-established mechanistic marker of oxidative injury [a). Similar to the previous experiments, we observed strong elevation of HO1 in TUNEL-positive cells after exposure to the compounds (b). The elevation was dose-dependent. However, dacarbazine killed melanoma cells without induction of HO1. We next examined whether caspase-activated DNase (CAD), a known mechanistic marker of caspase-mediated apoptosis [3, 4 and 5). These compounds induced the colocalization of CAD with TUNEL indicating that these chemicals were capable of inducing caspase-mediated apoptosis in addition to the caspase-independent one (c). For some reason, no dose dependency was observed, the colocalization was at the same level at 5 and 10 µM. Apparently, the CAD/TUNEL association had an upper limit and reached a plateau. Unlike our compounds, dacarbazine did not induce any association of CAD with TUNEL. This suggested dacarbazine did not induce caspase-mediated apoptosis at the used concentrations. Thus, our compounds had two mechanisms while dacarbazine had only one mechanism of cell death in this model. To determine whether there may be an association between DNA fragmentation measured by TUNEL assay, and immunocytochemistry (ICC) of EndoG, the marker of caspase-independent apoptosis [d). The increase was dose-dependent, and dacarbazine at 5 µM did not show any effect, while our compounds even at 5 µM showed the increase of EndoG-TUNEL association. Overall, the effect of our compounds in terms of EndoG involvement seemed to be similar to that of dacarbazine; all three compounds and dacarbazine induced caspase-independent apoptosis.Apoptosis is usually associated with profound DNA fragmentation that makes the cell death irreversible. DNA fragmentation could be due to the increasing of nuclear DNA access to apoptotic endonucleases and the induction of these enzymes ,19. Thisma cells a,c, and ompounds b,c. Contobserved d,e. Appae injury ,21. Becaompounds b. The elpoptosis ,23, is cdent one c. For sopoptosis , and thepoptosis ,26 knownpoptosis . The colin vitro, we applied the fluorescent TUNEL assay combined with quantitative ICC for three markers: HO1 for oxidative cell injury, CAD for caspase-mediated apoptosis and EndoG for caspase-independent apoptosis. Our TUNEL data showed that compounds are more toxic to melanoma cells than dacarbazine. Two of three compounds (4 and 5) induced oxidative injury as measured by the increase mean intensity of HO-1. However, none of the compounds or dacarbazine induced apoptotic endonucleases. This is not surprising because apoptotic endonucleases are commonly present in the cell in sufficient amounts to be able to destroy DNA upon cell death without induction (which most likely would not be possible in a dead cell).Thus, to determine the mechanism of action of our compounds on melanoma cells Because TUNEL assay measures DNA fragmentation by endonucleases, we use ICC for two endonucleases, EndoG and CAD, to determine the mode of cell death. Our experiments showed that our compounds induce both caspase-independent and caspase-dependent apoptosis, as well as oxidative injury to melanoma cells. Contrary to our compounds, the currently used anti-melanoma drug dacarbazine induced only caspase-independent apoptosis. No caspase-dependent apoptosis or oxidative injury was observed after exposure of melanoma cells to the drug. The fact that compounds use three cytotoxic mechanisms instead of one explains why our thiazole compounds are more effective.50 values at low single digit µM concentration. Some of these compounds are non-toxic or less toxic to healthy HFF1 cell lines. Mode of action has been determined by flow cytometry and TUNEL assay. Potent molecules were found to be apoptotic and DNA-fragmenting agents. The mode of action studies determined that our new compounds simultaneously use three cytotoxic mechanisms to kill melanoma cells. In vivo anti-melanoma and ADMET studies will be reported in due course.In conclusion, we have discovered potent anti-melanoma agents by screening a panel of 50 compounds and eight of these compounds showed potent anti-melanoma properties with GIClick here for additional data file.


Cyclopeptides or cyclic peptides are polypeptides formed by ring closing of terminal amino acids. A large number of natural cyclopeptides have been reported to be highly effective against different cancer cells, some of which are renowned for their clinical uses. Compared to linear peptides, cyclopeptides have absolute advantages of structural rigidity, biochemical stability, binding affinity as well as membrane permeability, which contribute greatly to their anticancer potency. Therefore, the discovery and development of natural cyclopeptides as anticancer agents remains attractive to academic researchers and pharmaceutical companies. Herein, we provide an overview of anticancer cyclopeptides that were discovered in the past 20 years. The present review mainly focuses on the anticancer efficacies, mechanisms of action and chemical structures of cyclopeptides with natural origins. Additionally, studies of the structure–activity relationship, total synthetic strategies as well as bioactivities of natural cyclopeptides are also included in this article. In conclusion, due to their characteristic structural features, natural cyclopeptides have great potential to be developed as anticancer agents. Indeed, they can also serve as excellent scaffolds for the synthesis of novel derivatives for combating cancerous pathologies. Cancer has become an enormous burden in society and a leading cause of death in the world. Apart from aging, the risk factors of modern daily life such as smoking, unhealthy diet and mental stress also play significant roles in the growing incidence of cancer . AccordiFrom ancient times, taking Chinese Medicine as an example, people have been familiar with medicinal properties of natural resources and the benefits of using them to treat diseases . Hence, Escherichia coli) . According to the SAR assessment results, the N-methyl alanine residue was not the key functional group for the cytotoxicity of apratoxins [Apratoxins are a novel family of cytotoxic cyclodepsipeptides, and they were isolated from cyanobacterium e models . Mechanie models . Liu et e models  discoverl Sec61α . Furtherl Sec61α  found thyl group . Likewisry claim . This SAtivities . Apratoxratoxins . Overall66) isolated from sea hare, aurilides B (150) and C (151) were obtained from cyanobacterium L. majuscula. Both aurilides B and C were cyclic depsipeptides with potent in vitro cytotoxic activities. In H460 cells and mouse neuroblastoma (neuro-2a) cells, the LC50 values of aurilide B were found to be 0.04 and 0.01 µM, respectively whereas the LC50 values of aurilide C were found to be 0.13 and 0.05 µM, respectively. Moreover, aurilide B was demonstrated to induce microfilament network disruption against A-10 smooth muscle cells with an IC50 value of 2.9 µM. In an in vivo experiment, aurilide B was shown to deliver net tumor cell killing activity in the NCI’s hollow fiber assay [Unlike aurilide (er assay . A quanter assay . Collect152–154) were cyclic depsipeptides isolated from L. majuscula with anti-proliferative effects against several cancer cell lines [50 values of lagunamides A were found ranging from 1.64 to 6.4 nM. Lagunamide B was only tested for its cytotoxicities against P388 and HCT8 cell lines; its IC50 values were determined to be 20.5 and 5.2 nM, respectively. Subsequent studies revealed that the cytotoxic effects of lagunamides A and B was highly associated with the induction of mitochondrial apoptosis [50 values varied from 2.1 nM to 24.2 nM against a panel of cancer cell lines such as P388, A549, PC3, HCT8 and SK-OV3 [Lagunamides A-C (ll lines . Tripathpoptosis . As for d SK-OV3 .L. majuscula was homodolastatin 16 (155), which was a homologue of the potent anticancer agent dolastatin 16. Compared to dolastatin 16, homodolastatin 16 only exhibited moderate activities against esophageal (WHCO1 and WHCO6) and cervical (ME180) cancer cell lines with IC50 values of 4.19, 9.85 and 8.09 µM, respectively [Another cyclic depsipeptide identified from ectively .156) was another cytotoxic cyclic depsipeptide derived from Guamanian strain of L. majuscula. Other than cytotoxic activities against KB cells (IC50 = 0.04 µM) and LoVo cells (IC50 = 0.72 µM), lyngbyabellin A also exhibited specific cytoskeletal-disrupting effects to microfilaments in A-10 cells at concentrations ranging from 0.01 to 7.24 µM [157), F (158) and H (159) were also isolated from L. majuscula. They exhibited cytotoxicity against H460 and KB cancer cells with IC50 or LC50 values ranging from 0.1 to 0.4 µM [160) from Moorea bouillonii, a rather new cytotoxic member of the lyngbyabellin class. This compound showed a potent anticancer potential against the CRC HCT116 cells with IC50 value of 40.9 nM [Lyngbyabellin A ( 7.24 µM . To furt 7.24 µM . Similaro 0.4 µM . In the  40.9 nM . Althoug161), another cyclic depsipeptide, was first isolated from L. majuscula. In the H460 cell line, palmyramide A displayed merely a weak cytotoxicity (IC50 = 39.7 µM). However, in neuro-2a cells, palmyramide A led to the blockage of the voltage-gated sodium channel with an IC50 value of 17.2 µM [Palmyramide A ( 17.2 µM . As a re162) and B (163) were two cyclic depsipeptides isolated from two distinct collections of marine cyanobacteria obtained from Papua New Guinea. Hoiamide A was a potent bioactive cyclic depsipeptide found in the assemblage of Lyngbya majuscula and Phormidium gracile [+ influx in mouse neocortical neurons [N-terminal kinase (JNK) and caspase signaling pathways [50 values of 11.2 and 8.3 µM, respectively [Hoiamides A  was a cyclic dodecapeptide derived from L. majuscula in Papua New Guinea [165), another novel cyclic peptide congener of wewakazole, from cyanobacterium Moorea peoducens. As cyanobactins, the most prominent bioactivity of the two compounds is cytotoxicity. When testing against H460 cells, wewakazole B (IC50 = 1.0 µM) was more cytotoxic than wewakazole (IC50 = 10 µM). Besides, wewakazole B also exhibited cytotoxicity against MCF7 cells with an IC50 value of 0.58 µM. Different from wewakazole, wewakazole B was inactive in siderophore assay [Wewakazole  was isolated from L. majuscula. It is a cyclic lipopeptide with moderate cytotoxicity against several cancer cell lines. Hectochlorin showed an average GI50 value of 5.1 µM in the NCI-60 screening assay; the colon, melanoma, ovarian and renal cell lines were the most sensitive to this compound [50 values of 0.86 and 1.20 µM, respectively [Hectochlorin , a cyclic depsipeptide found in the process of a cytotoxicity-guided fractionation, was isolated from L. majuscula. The cyclic DMMC exhibited potent selectivity and cytotoxicity against the cancer cell lines HCT116, H460 and MDA-MB-435 with IC50 values of 0.02, 0.063 and 0.22 µM, respectively. Furthermore, a ring-opened form of DMMC was produced by means of base hydrolysis. Remarkably, the linear form of DMMC shared the same potency of cytotoxicity against the same panel of cancer cell lines . When acting against A10 cells, both cyclic and linear DMMCs disrupted the cellular microfilament networks and caused dramatic alterations in cell morphology, which were shown as the result of cellular apoptosis. Overall, these biological data indicated the potential anticancer efficacy of both cyclic and linear forms of DMMC [Desmethoxymajusculamide C (DMMC) ( of DMMC . The sig168), another cyclodepsipeptide isolated from L. majuscula, was first discovered in Singapore. The compound displayed in vitro cytotoxicities against human lymphoblastic leukemia (MOLT-4) and MCF7 cells with IC50 values of 32 nM and 4.0 µM, respectively [169) and C (170). Both compounds exhibited cytotoxicities when tested against MOLT-4  and MCF7  cells [Hantupeptin A (ectively . A subsey) cells . Furthery) cells ,250.L. majuscula were designated as laxaphycins A (171) and B (172). Laxaphycin B showed significant cytotoxicities against the CCRF-CEM drug-sensitive human leukemic lymphoblasts (IC50 = 1.11 µM), as well as the vinblastine-resistant subline overexpressing Pgp CEM/VLB100 (IC50 = 1.02 µM) and CEM/VM-1 subline with DNA topoisomerase II alteration (IC50 = 1.37 µM). However, laxaphycin A was found to be inactive towards these cell lines at 20 µM [50 values ranging from 0.19 to 1.0 µM. Furthermore, obvious biological synergism was observed on DLD1  and MDR leukemic cells in the presence of laxaphycins A and B. Similar to the compounds mentioned previously, laxaphycins B2 (173) and B3 (174) were also isolated from the same assemblage of the cyanobacteria. These two novel cyclic peptides, displayed merely moderate cytotoxicities against the three leukemic cell lines above [175) was a new member of B-type laxaphycins, and it was discovered from the marine cyanobacterium Hormothamnion enteromorphoides. This compound yielded growth-inhibitory activity against HCT116 (IC50 = 1.7 µM); synergistic effect was obtained when it was used in combination with laxaphycin A [Two cyclic peptides isolated from at 20 µM . Subseques above . Laxaphyphycin A .176) was a cytotoxic cyclic depsipeptide isolated in L. confervoides. From the MTT assay results, we noticed that this compound exhibited cytotoxicity against KB and LoVo cells with IC50 values of 0.97 and 5.24 µM, respectively [β-amino acid was an essential moiety for the bioactivities of obyanamide and its analogs [Obyanamide  was a macrocyclic depsipeptide with anticancer potential isolated from L. confervoides. Anti-proliferative activities of grasssypeptolide A were investigated in the U2OS (IC50 = 2.2 µM), HeLa (IC50 = 1.0 µM), HT29 (IC50 = 1.5 µM) and IMR-32 (IC50 = 4.2µM) cancer cell lines [177–179). The in vitro anti-proliferative effects of these compounds were evaluated in HT29  and HeLa  cells. According to the cell viability assay results, grassypeptolide C was 16-23 times more potent than grassypeptolide A. Moreover, both grasspeptolides A and C could induce cell cycle arrest at the G1 phase at relatively low concentrations ; they could also induce the G2/M phase arrest at higher concentrations . These two compounds have the capability to bind to metal ions such as Cu2+ and Zn2+, which may denote a partial mechanism of their cytotoxicities [180) and E (181), two novel members of the family, were isolated from the marine cyanobacterium Leptolyngbya sp. These two compounds were cytotoxic to HeLa cells  and mouse neuro-2a blastoma cells  [Grassypeptolide A (ll lines . Subsequxicities . The stuxicities . Grassypctively) . To prom182), a cyclic depsipeptide extracted from a bioassay-guided fractionation, was confirmed as a potent cytotoxin isolated from Lyngbya sp. in Palau. When cultured with KB cells, palauamide yielded significant cytotoxicity with an IC50 value of 13 nM [Palauamide  was a cyclic depsipeptide that was first isolated from the marine filamentous cyanobacterium Leptolyngbya sp. in Coiba National Park, Panama. The preliminary biological activity screening showed that this compound displayed potent cytotoxicity against NCI-H460 and mouse neuro-2a cells with LC50 < 17.9 nM. The evaluation against the NCI’s in vitro 60 cancer cell line panel highlighted its remarkable anti-proliferative response. From the flow cytometric analysis, coibamide A was shown to induce cell cycle arrest at the G1 phase [3 to 200-300 mm3. However, different degrees of weight loss were observed in the animal experiment. Such result implicated that targeted delivery and toxicity profile should be improved for drug development [Coibamide A (G1 phase . SubsequG1 phase  demonstrelopment .184–188) were five β-amino acid-containing cyclopeptides derived from the marine cyanobacterium Lyngbya sp. in Palau. These ulongamides exhibited moderate in vitro cytotoxicity against KB and LoVo cells with IC50 values around 1 and 5 µM [Ulongamides A-E , another cytotoxic cyclopeptide, was isolated from Lyngbya sp. in Palau. This compound yielded cytotoxicity against KB cells with an IC50 value of 0.63 µM [Ulongapeptin ( 0.63 µM . No furtOkeania sp. is the origin of the cytotoxic cyclodepsipeptide odoamide (190). As another new member of aurilide class, odoamide has been reported with a significant cytotoxicity against HeLa S3 cells (IC50 = 26.3 nM) and a moderate toxicity to brine shrimp [The marine cyanobacterium e shrimp . From the shrimp .191), a cyclic depsipeptide isolated from cyanobacterium Nostoc sp., was first discovered as an antifungal agent two decades ago. Subsequent studies indicated that cryptophycin 1 was also an anti-proliferative agent with significant antitumor activities [50 = 4 pM), SKOV3 (IC50 = 7 pM) and MCF7 (IC50 = 16 pM). Some recent reports demonstrated that cryptophycin 1 provided an anticancer potency ~100-fold higher than the mainstay anticancer drug paclitaxel; therefore, a great interest was immediately prompted to investigate the mechanism of action of this compound [Cryptophycin 1  is a cyclic depsipeptide isolated from the cyanobacterium of Symploca sp. This compound was found to have good selectivity against different types of cancer cells. For instance, largazole was less cytotoxicy to the nontransformed murine mammary epithelial MDA-MB-231 cells , but it was more susceptible to the invasive transformed human mammary epithelial (NmuMG) cells . Similarly, this cyclopeptide displayed higher inhibitory effect against U2OS transformed fibroblastic osteosarcoma  than NIH3T3 nontransformed fibroblasts  [50 values of 0.08 µM and 0.32 µM, respectively [Kip1 and telomeric repeat-binding factor 1 (TRF1) in vitro [Largazole ( > 8 µM) . Based o > 8 µM) . In the ectively . The in ectively . It is win vitro . In addiin vitro . While sSymploca sp. From Papua New Guinea led to the isolation of a novel cyclopeptide—symplocamide A (193). This compound showed potent cytotoxicity against H460 and neuro-2a cells with IC50 values of 40 nM and 29 nM, respectively. In fact, symplocamide A shares a structure very similar to protease inhibitors; thus, it can be served as a potent chymotrypsin inhibitor (IC50 = 0.38 µM). The role of protease inhibition in cancer therapy is worth to be studied. On the other hand, symplocamide A was reported to show moderate anti-parasite activities against malaria, Chagas disease and leishmaniasis [Studies on a cyanobacterium of maniasis . Solid pmaniasis .Symploca sp. in Palau, and they were named tasipeptins A (194) and B (195). These two compounds exhibited cytotoxicities against KB cells with IC50 values of 0.93 and 0.82 µM, respectively [Two other cyclic depsipeptides had been isolated from ectively . Due to ectively . It is bSymploca cf. hydnoides from Guam led to the isolation of a series of cyclic depsipeptides, veraguamides A-G (196–202). The results of several cell viability assays showed that veraguamides A-G displayed weak to moderate cytotoxicities against HT29 and HeLa cells with IC50 values varying from 0.84 to 49 µM and 0.54 to 49 µM, respectively. From the SAR assessments, the seven semi-synthetic analogs of veraguamide G explicitly denoted the sensitive positions of veraguamides that were responsible for their cytotoxic activities [Cytotoxicity-directed fractionation of cyanobacterium tivities .203), a novel cyclic depsipeptide containing four amino acid units, was discovered in a bioassay-guided investigation of Moorea producens, a species of filamentous cyanobacteria collected from Florida, USA. This compound displayed cytotoxic activities against H460 and mouse neuro-2a neuroblastoma cells with the EC50 values of around 1.89 × 10−5 µM [Floridamide ( 10−5 µM .204) was a highly toxic cyclic depsipeptide found in the cyanobacterium Rivularia sp. near the island of Vieques. It was found to be strongly cytotoxic against H460 cells with an IC50 value of 60 nM. As a member of the “kulolide superfamily”, the biological properties of viequeamied A were reported to be highly divergent [Viequeamide A (ivergent . The totivergent .Fungi are eukaryotic organisms with worldwide distribution and different habitats. It is estimated that biodiversity of fungi is between 2.2 to 3.8 million species around the world . Moreove205) was a cyclic tetrapeptide isolated from the fungi of Tolypocladium sp. obtained from the Great Lakes. The compound displayed predominant anti-proliferative and cytotoxic activities toward MIA PaCa-2 cells with GI50 and LC50 values of 5.3 and 22 nM, respectively. Moreover, as a potent HDAC inhibitor, 1-alaninechlamydocin induced apoptosis and cell cycle arrest at phase G2/M as well. HDAC inhibitory activity also primarily contributed to in vitro bioactivities of the compound [1-Alaninechlamydocin  showed moderate cytotoxicity. The compound exhibited anti-proliferative effect to K562 cells with the GI50 value of 6.8 µM and cytotoxicity to HeLa cells with CC50 value of 16.68 µM. However, human umbilical vein endothelial cells (HUVEC) was also sensitive to pseudoxylallemycin C (GI50 = 6.96 µM), which demonstrated low specificity of the compound [compound .207) and G (208) were two new members of cycloaspeptides—the ABA-containing cyclic pentapeptides discovered in different fungi. These two compounds were derived from the fungus Isaria farinosa, an inhabitant of Cordyceps sinensis obtained from Linzhi, Tibet. They showed inhibitory activities toward MCF7 cells with respective GI50 values of 18.7 and 15.2 µM respectively, which were comparable to the positive control 5FU (GI50 = 15 µM) [Cycloaspeptides F (= 15 µM) .1 (MA1) (209), a cyclopentapeptide with versatile biological activities, was originally isolated from the terrestrial plant fungus Aspergillus niger. Liu et al. investigated its anticancer mechanism of action in human prostate cancer PC3 (IC50 = 130 nM) aand LNCaP (IC50 = 90 nM) cell lines. Coexistence of apoptosis, necrosis and autophagy were all induced by mitochondrial damage, which illustrated the mechanism of MA1-mediated cell death in prostate cancer cells. In addition, stimulation of the AMP-activated serine/threonine protein kinase/mammalian target-of-rapamycin (AMPK/mTOR) pathway by adding oxidative stress or decreasing ATP could lead to MA1-mediated autophagy [A. tubingenesis also yielded MA1 with significant cytotoxicity against HeLa (IC50 = 9.48 nM) and P388 (IC50 = 132.9 nM) cell lines [1 is an early-discovered cyclic peptide with diverse biological properties and sufficient studies have demonstrated its bioactivities as well as mechanism of action. However, for further promotion to clinical stage, more supporting data in animal models shall be carried out. Another new member of the malformin class, designated as malformin E (210), was isolated from the endophytic fungus A. tamarii collected from the roots of Ficus carica. The compound also showed in vitro cytotoxic effects on MCF7 and A549 cells, with IC50 values of 0.65 and 2.42 µM, respectively. Moreover, malformin E also exhibited antimicrobial activities against several strains of bacteria and fungi [Malformin Autophagy . In humautophagy . Recentlll lines . Malformnd fungi .228), neo-N-methylsansalvamide (211), was isolated from the fungus Fusarium solani KCCM90040 derived from the potato in Korea. The new cyclic pentadepsipeptide was able to inhibit in vitro cell growth of A549, SK-OV-3, SK-MEL-2 (human melanoma) and MES-SA (human uterine sarcoma) cells with EC50 values ranging from 10.0 to 14.74 µM [N-methylsansalvamide and paclitaxel were also observed when acting against MES-SA, HCT15  and two multidrug resistance sublines (MES-SA/DX5 (human uterine corpus sarcoma) and HCT15/CL05). The EC50 values of paclitaxel added with 3 µM neo-N-methylsansalvamide were significantly decreased by several orders of magnitude [Another new analog of sansalvamide A (14.74 µM . Moreoveagnitude . As a re212), a cyclic nonapeptide, was isolated from Cyclindrocarpon sp. strain A101-96 during the period of a screening of new metabolites with cytotoxic effects from fungal extracts. Six cancer cell lines were assayed for its anticancer potential, while the compound only showed moderate cytotoxicity to colorectal adenocarcinoma (COLO-320), human leukemia (HL-60), L1210 and human T lymphocyte Jurkat cell lines with IC50 value around 11 µM. Neither antimicrobial or nematicidal effect was observed for cylindrocyclin A [Cylindrocyclin A  and B (214). Both compounds are octadecacyclopeptides with natural cyclic β hairpins. Gymnopeptides A and B displayed significant anti-proliferative activities against HeLa, A431 (human epidermoid carcinoma), T47D, MCF7 and MDA-MB-231 cells with IC50 values ranging from 18.0 to 88.4 nM and 14.0 to 44.3 nM, respectively [Studies on the mushroom ectively . Due to ectively . As a re215), a cyclic hexapeptide derived from the fungus Diaporthe toxica, was also an N-methylated derivative of the mycotoxin phomopsin A. The initial in vitro screening of cytotoxicity revealed its impact on viability and vitability of HepG2 cells (IC50 = 6.1 µM) [Phomopsin F ( 6.1 µM) .216–219), were purified from a basidiomycete identified as Pterula species. Pterides I and II showed remarkable cytotoxicity against P388 cell line with IC50 values of 41 and 40 nM, respectively. Nevertheless, compared to the first two compounds, pteratides III and IV were much less active  due to their much different structural discrepancy to pteratides I and II [Four cytotoxic cyclodepsipeptides, pteratides I-IV (I and II . Practic220), with entire composition of non-protein amino acids, was first isolated from the marine-derived fungus Aspergillus terreus SCSGAF0162  and B (222) were two bioactive cycloheptapeptides previously isolated from two different insect pathogenic fungi Cordyceps sp. BCC 1788 and 16173, respectively. The two cyclic peptides displayed anti-proliferative activities against KB, BC (human lymphoma), NCI-H187 and Vero  cells with IC50 values of cordyheptapeptide A measured as 0.78, 0.2, 0.18 and 14 µM, respectively, the IC50 values of cordyheptapeptide B as 2.0, 0.66, 3.1 and 1.6 µM, respectively. In addition, cordyheptapeptide A also exhibited moderate antimalarial activities [50 = 10.6 and 7.4 µM, respectively) and Ehrlich’s ascites carcinoma (EAC)  cell lines [223) and E (224), from a marine-derived fungus Acremonium persicinum in 2012. Cordyheptapeptide C displayed cytotoxicity against SF-268 and MCF7 with IC50 values of 3.7 and 3.0 μM, respectively. And the other one showed similar cytotoxicity against SF-268, MCF7 and NCI-H460 cell lines  [Cordyheptapeptides A  . However225) and B (226) were two cyclic heptapeptides extracted from the culture broth of the marine fungus Scytalidium sp. from the Bahamas. When incubated with HCT116 cell line, the two compounds displayed in vitro cytotoxicity with IC50 values of 2.7 and 11.0 µM, respectively. Tested in the NCI 60 cell line panel, scytalidamides A and B exhibited moderate cytotoxicity with mean GI50 values of 7.9 and 4.1 µM, respectively. The screening test also demonstrated the most sensitive cell lines were MOLT-4 (IC50 = 3.0 µM) for scytalidamide A and UACC-257 (IC50 = 3.0 µM) for scytalidamide B [Scytalidamides A (damide B .227), a cyclic pentadepsipeptide produced by the marine-derived fungus Zygosporium masonii, was shown to possess potent anticancer properties. Evaluated in the NCI cell line assays (median GI50 = 9.1 µM), the compound yielded significant in vitro cytotoxicity against central nervous system (CNS) cancer cell line SF-268 (IC50 = 6.5 nM) and renal cancer cell line RXF393 (IC50 ≤ 5.0 nM) with high selectivity [50 values between the natural and synthetic cyclic peptides when acting against SF-268 [Zygosporamide (ectivity . The tott SF-268 .228), a natural cyclic depsipeptide, was isolated first from a marine fungus of Fusarium sp. living on the seagrass Halodule wrightii. The compound showed cytotoxicity against several cancer cells lines, including HCT116, COLO 205 and SK-MEL-2 cells with IC50 values of 9.7, 3.46 and 5.84 μM, respectively [Sansalvamide A  and B (230) were two novel cyclodepsipeptides derived from the fungus Aspergillus clavatus. Xenograpsus testudinatus, a kind of hydrothermal vent crab, is the habitat where they found the fungus. Jiang et al. determined that both compounds suppressed the proliferation of HCC (hepatocellular carcinoma) cell lines  in a dose-dependent as well as time-dependent manner, with IC50 values around 32 μM. Compared with human normal hepatocyte, HCC cell lines, especially HepG2 cells, were more sensitive in clavatustides-induced cell proliferation suppression. Their proliferation inhibitory property was due to suppression of G1/S phase transition [Clavatustides A  was isolated from the fungus er 20 μM .232) and B (233), were isolated from the fungus Scopulariopsis brevicaulis, which was found in the marine sponge Tethya aurantium. Both compounds significantly inhibited viability of several cancer cell lines including Colo357 (pancreatic adenosquamous carcinoma), Panc89 (pancreatic tumor) and HT29 cells at a concentration of 14.87 μM [Two cyclodepsipeptides, scopularides A (14.87 μM . However234) was first isolated from the marine fungus Clonostachys sp. ESNA-A009. It was highly active against several cancer cell lines including LNCaP, SKBR3, HT29 and HeLa with the GI50 values on the order of 10−8 M [Leishmania [N-methylation on the amino acids of IB-01212 were found to enhance its cytotoxic effect, which were corroborating evidence for further discovery and synthesis of new analogs for anticancer drug development [The cytotoxic cyclodepsipeptide IB-01212  and B (236) were cyclopeptides isolated from the marine fungus Microsporum cf. gypseum in bryozoan Bugula sp. from Virgin Islands in the US. Both were shown to be potent HDAC inhibitors and demonstrated cytotoxicity especially against HCT116 cell line, with IC50 values of 1.12 and 15.82 μM, respectively [Microsporins A (ectively . To exteectively ,334.This review included the recently discovered natural anticancer cyclopeptides, which have been summarized and categorized according to their origins. An abundant number of cyclopeptides with diverging chemical and biological characteristics can be obtained from the aqueous and terrestrial portions of our nature. Cyclopeptides derived from natural sources have drawn great attention due to their high therapeutic potential for drug development. Today, many studies have shown that natural cyclopeptides are effective in the treatment and preventive measures of cancerous pathologies. Other biological activities such as antiviral, antibacterial and antimalarial effects have also been extensively observed whilst some are considered promising. Hence, detailed mechanism studies and structural modification of cyclopeptides are desperately needed for the development of cyclopeptide as therapeutic drug candidates. Nonetheless, the current research studies on the anticancer effects of natural cyclopeptides are just the tip of the iceberg. The natural cyclopeptides are undoubtedly a rich source as bioactive compound leads gifted from nature to benefit human welfare.