BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either  DLBCL remains fatal in over a third of patients; the one major step forward has been the addition of the CD20-specific antibody rituximab to standard chemotherapy almost two decades ago [MYD88 and CD79B mutations and BCL2 gains, extranodal manifestations, a genetic signature of aberrant somatic hypermutation driven by activation-induced cytidine deaminase activity and a dismal prognosis; the other subtype is characterized by NOTCH2 and BCL6 mutations and structural aberrations, respectively, and the associated downstream transcriptional signatures, a presumably extrafollicular origin more reminiscent of marginal zone lymphoma, and a comparatively superior prognosis [BCL2 to the IgH enhancer in combination with frequent mutations in the chromatin modifers KMT2D, CREBBP, and EZH2, and inactivating PTEN mutations, bears similarities to the genetic landscape of follicular lymphoma and features a poor prognosis, whereas the other is a relatively low-risk subtype with mutations in PI3K, JAK/STAT, and BRAF pathway components [Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of the mature B-cell that may arise ades ago , 2. CAR ades ago  and the ades ago , 5. DLBCades ago . One of ades ago . Whereasades ago , 9, moreades ago  or five ades ago  differenrognosis , 11. Simmponents , 11.BCL2 amplifications on the one hand, and a constitutively active B-cell receptor/Bruton’s tyrosine kinase/NF-κB signaling axis on the other.The substantial intertumoral heterogeneity of DLBCL suggests that individual tumors will exhibit vastly different susceptibilities to drugs- both approved compounds and drugs in clinical development. Surprisingly little attention has been paid in the field to differential drug responses in DLBCL and to the possible use of drug response profiling as a tool for guiding treatment decisions. Here, we report on a screen conducted on 19 DLBCL and other lymphoma cell lines, of drug susceptibility to a selection of 126 compounds that are approved for clinical use. Our results suggest that the Bcl-2 inhibitor venetoclax, in combination with the BTK inhibitor ibrutinib, is highly effective in the treatment of a particularly aggressive DLBCL subtype exhibiting We used a panel of 13 previously described DLBCL –14, fourh;IL-3/GM-CSFh;hSIRPAtg;TPOh;Rag2−γc− (MISTRG) [h (MISTRG6) [7 cells in 100 µl PBS) into 6- to 8-week-old mixed-gender MISTRG or MISTRG6 mice. Spleens and bone marrow from hind legs were harvested for analysis at the study endpoint of orthotopic models. All animal studies were reviewed and approved by the Zurich Cantonal Veterinary Office . For patient‐derived xenograft transplantation, primary DLBCL cells (obtained from the Clinic of Hematology–Oncology at the University of Zürich) were transplanted by i.v. injection (1 × 106 cells) to MISTRG6 mice as previously described [M-CSF(MISTRG)  and MISTMISTRG6)  were obtescribed . EthicalAll statistical analyses were performed using GraphPad Prism software. Graphs represent means plus standard deviation of at least two independent experiments, and statistical analysis was performed using two-tailed Mann–Whitney test, or one-way ANOVA  or by non-parametric ANOVA  with Tukey’s multiple comparisons correction. For animal experiments, studies were repeated and multiple studies were pooled where one experiment alone was not sufficient to reach statistically significant conclusions.We systematically assessed the sensitivities of 19 lymphoma cell lines  to 126 FDA-approved compounds, which were chosen based on their activity on pathways that are known to be deregulated in hematopoietic malignancies , Bcl-2 and the AKT isoforms 1–3. Interestingly, only cell lines that were identified in the screen to be sensitive to AKT inhibition were, if subjected to PTEN, a tumor suppressor and negative regulator of the AKT signaling pathway, as the cell lines harboring a mutant PTEN allele [We have previously subjected the DLBCL cell lines included in the drug screen described above to an in depth characterization of their mutational landscapes . InteresN allele  were amoBCL2 to IgH gene enhancer elements and affects ~20% of GCB-DLBCL but is rare in ABC-DLBCL, or by BCL2 amplification in ABC-DLBCL [BCL2 amplifications; these are the cell lines with the highest Bcl-2 expression  translocation, which juxtaposes BC-DLBCL . Of six ion Fig. . Of the ion Fig. . The ABCels Fig. . Bcl-2 eels Fig. . Of 206 els Fig. . Venetocels Fig. . The Bclype Fig. .Fig. 2ThBCL2 amplification and/or translocation is required, but not sufficient for responses to venetoclax in vitro and in vivo.We next asked whether the differential susceptibility to venetoclax can be recapitulated in vivo in a suitable xenotransplantation model of DLBCL. To this end, luciferase-expressing RIVA, U2932 and HBL-1 cells were injected into genetically humanized mice . All thrL  transcript levels were highest in the «BN2» genetic subtype  expressed very high levels of Bcl-XL Fig. . Mcl-1 aype Fig. , which hype Fig. , indicatnts Fig. . An invents Fig. . Interesnts Fig. . In summBCL2 alleles and no or minimal Bcl-2 expression were totally resistant to venetoclax, also in the combination with ibrutinib ; (2) cell lines with moderate Bcl-2 expression due to the t translocation were primarily resistant to venetoclax, but could be sensitized by ibrutinib ; (3) cell lines with high Bcl-2 expression either responded (RIVA) or not  to single-agent venetoclax, but were all readily killed by the combination treatment  in the resistant OCI-LY3 cell line under in vitro exposure to ibrutinib (data not shown), this number was much higher for the sensitive cell lines . Whereas the expression of BCL2 and MCL1 was not found to be controlled by ibrutinib in this unbiased analysis, neither in vitro nor in vivo, two other anti-apoptotic Bcl-2 family members, BCL2L1 (encoding Bcl-XL) and BCL2A1 (encoding Bcl-2 related protein A1) were strongly downregulated in at least three of the four examined conditions  and one resistant (OCI-LY3) cell line to ibrutinib exposure and performed bulk RNA sequencing after 6 h of exposure in vitro or two weeks of continuous treatment in vivo. Principle component analysis  co-occurring MYD88/CD79 mutations and ABC-DLBCL origin, in which Bcl-2 overexpression is driven by gene amplification, or alternatively, featuring (2) BCL2 translocations, co-occurring activating EZH mutations and a GCB-DLBCL cellular origin. Examples of both DLBCL subtypes were represented among our panel of DLBCL cell lines and could be used to confirm the genetic dependence of these - but not of Bcl-2low - cell lines on Bcl-2 expression in competition experiments, for which the BCL2 locus was selectively ablated by genome editing. The same cell lines, equipped with luciferase to allow for their tracking by IVIS, responded to venetoclax exposure in vitro and in vivo, albeit in most instances only in combination with ibrutinib or the PI3K inhibitor duvelisib. Bcl-2low cell lines and primary tumors from DLBCL patients tend to alternatively express Mcl-1, which is inversely correlated with Bcl-2 at the transcript level; the DLBCL subtype featuring NOTCH2 mutations and BCL6 translocations  is parted “BN2”  to selecint and Bcl-2hi cell lines in vitro, but also led to faster and more complete tumor regression in vivo of cell lines with primary as well as acquired resistance to venetoclax. All available in vivo data thus lend support to the addition of ibrutinib to the venetoclax regimen in relapsed or refractory patients with the intention of overcoming primary resistance and delaying, overcoming or preventing acquired resistance to venetoclax. Simple immunohistochemical analysis of Bcl-2 and of phosphorylated or nuclear P65 as a marker of constitutive NF-κB activation are potentially useful indicators of efficacy of the combinatorial treatment; six of 39 patients in our DLBCL patient cohort that were evaluable for both markers had evidence of both high Bcl-2 expression and of nuclear distribution of NF-κB P65.To systematically search for additional resistance mechanisms, we conducted a combinatorial screen in which 62 FDA-approved compounds were assessed for their ability to sensitize DLBCL cells to venetoclax-induced toxicity. Two categories of drugs emerged from this screen, which target BTK on the one hand , and the phosphatidylinositol 3-kinase  on the other. We focused our in vivo studies mostly on the venetoclax/ibrutinib combination as ibrutinib is safe , but unfPreclinical evidence for the synergy between venetoclax and ibrutinib has been reported previously in studies that attempted to overcome ibrutinib resistance in follicular lymphoma and DLBCL , and othL and Bcl-2A1 as alternative Bcl-2 family members that can substitute for Bcl-2 unless transcriptionally downregulated by BTK inhibition. Our results are in line with the conclusion that two independent survival pathways, one acting at the mitochondrial membrane and preventing the pro-apoptotic activities of Bcl family proteins such as Bak, Bax and Bid, and the other acting at endolysosomal membranes relaying survival signals via the multiprotein supercomplex formed by MYD88, TLR9 and the BCR [The mechanism of ibrutinib/venetoclax synergy remains a matter of debate. We found no evidence of differential regulation of Bcl-2 or Mcl-1 under ibrutinib, as shown previously by others . Rather, the BCR , have tosuppl. table 1suppl. table 2suppl. table 3suppl. table 4Supplemental figures and methods


Marine natural products are promising candidates for lead pharmacological compounds to treat diseases that plague humans, including cancer. Cancer is a life-threatening disorder that has been difficult to overcome. It is a long-term illness that affects both young and old people. In recent years, significant attempts have been made to identify new anticancer drugs, as the existing drugs have been useless due to resistance of the malignant cells. Natural products derived from marine sources have been tested for their anticancer activity using a variety of cancer cell lines derived from humans and other sources, some of which have already been approved for clinical use, while some others are still being tested. These compounds can assault cancer cells via a variety of mechanisms, but certain cancer cells are resistant to them. As a result, the goal of this review was to look into the anticancer potential of marine natural products or their derivatives that were isolated from January 2019 to March 2020, in cancer cell lines, with a focus on the class and type of isolated compounds, source and location of isolation, cancer cell line type, and potency (IC For decades, marine natural products have been sources of bioactive compounds with remarkable biological activities. These include anticancer, antimicrobial, antidiabetic, anti-inflammatory, antiparasitic, antihypertensive, and antioxidant activities. They can be traced to the effects of numerous marine compounds from different classes of constituents such as alkaloids, terpenoids, steroids, flavonoids, peptides, proteins, polyphenols, carotenoids, vitamins, tocopherols, tocotrienols, peptides, carbohydrates, etc. Not only marine compounds, but also terrestrial plant constituents have anticancer properties ,2,3,4,5,Compounds with an outstanding activity profile in terms of efficacy and potency are chosen for further optimization and subsequent development into new drugs. Screening compounds for anticancer activity is time consuming. However, in accordance with the with the NCI’s disease-oriented drug screening approach for anticancer agents, the testing of compounds for anticancer activity involves the use of cancer cell lines or xenografts to achieve the desired effect. There has recently been a paradigm shift away from cytotoxic chemotherapy and towards target drug discovery. Target drug discovery entails the identification of molecular targets and the development of drugs that target those targets by the application of high-throughput molecular screening of hits. The selected hits are then subjected to biological activity confirmation in a living cell, followed by a determination of the new compound’s specificity using adequate cancer cell lines containing target receptors . Cancer is synonymous with a neoplasm or malignant tumor. It is a deadly disease that lacks growth control and has the ability to invade neighboring tissues and organs of the body from its origin and eventually destroys them. Cancer is a genetic disease caused by alterations to genes that control the way cells function, grow, and divide. According to the National Cancer Institute (NCI), genetic changes can cause cancer. Cancer-causing genetic changes can be inherited from parents or develop during a person’s lifetime as a result of cell division errors or DNA damage caused by exposures to certain environmental factors . Cancer-Tumors are classified as benign or malignant. Benign tumors are non-aggressive in nature because they are localized to a particular site in the body and can surgically be removed while malignant tumors spread from one location to another location, thereby making them aggressive and devastating. Sarcomas are malignant tumors that arise from solid mesenchymal tissues; for example, fibrosarcoma is cancer of fibrous tissue, and lymphomas or leukemias arise from the blood mesenchymal cells . CarcinoThe cell cycle is a normal physiological process that occurs in distinct stages in the cell to enable the proliferation of cell growth, development, and maintenance of the species. Cell proliferation and progression in normal cells are under tight genetic control. For cancer to develop, there must be a change in the genetic machinery that controls cell proliferation and eventual progression of cell cycle stages, which is the hallmark of cancer growth and sustenance .Chemotherapeutic agents, targeted therapy/immunotherapy, radiation, surgery, or a combination of these treatment approaches can all be used to treat cancer, depending on the cancer stage. Chemotherapy is the center of our attention in this review. Cytotoxic agents inhibit or kill cancer cells by diverse mechanisms, some of which are well understood while others are not. Some drugs are designed to target a cancer cell’s pathophysiological survival processes, while others function as suicidal agents, altering the proliferation and metabolic activities of the cancer cells. Cancerous cells and normal cells share similar genetic machinery for growth and development at the cellular level. Targeting the genetic architecture of malignant cells has resulted in significant advances in the treatment of various malignancies. Some anticancer medications work by affecting cell division in rapidly diving cells, making them antiproliferative. These medications are used to stop cancer cells from progressing through the stages of the cell cycle. Some medications cause a cell arrest that is specific to a particular cell cycle stage whereas others are not. Many of them are most effective in the S-phase, causing DNA damage and triggering programmed cell death. Some block DNA synthesis either by inhibiting purine/pyrimidine synthesis or DNA polymerase inhibition. Other drugs cause direct damage by breaking DNA chains, cross-linking DNA strands, and inhibiting depolarization, while others inhibit transduction of DNA to RNA, damage mitotic spindles, or inhibit the topoisomerase enzyme .Anticancer drugs used clinically based on their mechanism of action are classified into cell cycle non-specific (CCNSAs) and cell cycle specific agents (CCSAs). CCNSAs are drugs that are active throughout the cell cycle, e.g.,1. Alkylating agents: nitrogen mustards , nitrosoureas , tetrazines , and aziridines  that deposit an alkyl group on DNA, forming a covalent bond with the nucleophilic nitrogen at position 7(N7) of guanidine or N1 and N3 of adenine and N3 of cytosine residues in DNA. This leads to an abnormal base pairing and DNA strand breakage, blockade of nucleic acid biosynthesis, direct destruction of DNA and inhibition of DNA reproduction, interference of transcription, and blockade of RNA synthesis with interference of protein synthesis and function, leading to eventual apoptosis ,20,21, a2. Antibiotics: e.g., actinomycin that inhibits transcription by binding DNA at the transcription initiation complex and preventing elongation of the RNA chain by RNA polymerase .CCSAs act at a specific phase of the cell cycle: (a) S-phase-specific drugs:(i) Antimetabolites  act by inhibiting the enzyme involved in the synthesis of DNA or are incorporated as toxic analogues in the backbone of DNA during synthesis, leading to prevention of mitosis and DNA breakage ,21 (ii) Topoisomerase inhibitors (two types—topoisomerase I and II). Topoisomerase I inhibitors include irinotecan and topotecan. Topoisomerase II is further divided into topoisomerase II poisons that target topoisomerase–DNA complexes, preventing DNA replication and transcription, thus, causing DNA strand breakage, and consequent apoptosis. Examples of topoisomerase II poisons are etoposide, doxorubicin, mitoxantrone, and teniposide . The secVinca alkaloids by binding tubulin and preventing microtubule assembly  cytochalasans (Cpds. 4–9.2 µM  42]..28–43), 45) and didiscorhabdin (Cpd. 46), were isolated from Latrunculia biformis, a sponge from the Weddell Sea (Antarctica). As novel compounds, a unique C-N bridge (C-1/N-13) between discorhabdin monomers was present in them, with Cpd. 45 being the first trimeric discorhabdin molecule isolated from the natural environment. Additionally, this compound was cytotoxic to human colon cancer cell line HCT 116 at 0.31 µM IC50. It equally inhibited the non-cancerous human keratinocyte cell line HaCaT with a similar IC50 value (IC50 = 0.92 μM), thus, suggesting its general toxicity and low selectivity for cancer cells; the structure is in Following the LC-MS/MS molecular networking-based metabolomics and cytotoxic activity-guided study, two new discorhabdin-type alkaloids, tridiscorhabdin (Cpd. 47), 7-methoxy-7’-hydroxyepicoccin G (Cpd. 48), 8’-acetoxyepicoccin D (Cpd. 49), 7’-demethoxyrostratin C (50), a pair of new enantiomeric diketopiperazines, (+/−)-5-hydroxydiphenylalazine A , and five known compounds (Cpds. 53–57), were isolated from deep-sea fungus Epicoccum nigrum SD-388 extract. Cpds. 50 and 57 had good antiproliferative activity against Huh7.5 with the respective IC50 values of 9.52 and 4.88 μM. Their cytotoxic activities can be compared to that of sorafenib (IC50 = 8.2), the positive control, although compound 10 was more potent. The disulfide bridge at C-2/C-2’ seems to be crucial for the anticancer activity ..50 valueBurkholderia sp. NBF227. A series of novel beta-carboline analogues, 9a-p (Cpds. 69–84) derived from pityriacitrin, were tested for anticancer activity against the following human cancer cell lines: SGC-7901, A875, HepG2, and MARC145. Some of these beta-carboline derivatives exhibited moderate to high cytotoxic activities. Compound 9o (Cpd. 83) with a sulfonyl group had the highest inhibitory activity against all the cell lines with the IC50 values of 6.82, 8.43, 7.69, and 7.19 µM, respectively. The substitution of the amide scaffold attached on the compounds was carried out with the following substituents: a phenylethylamine for compounds 9a–f (Cpds. 69–74), or a benzylamine for compounds 9g–l (Cpds. 75–80), or an arylamine for compounds 9m–n , and that did not yield analogues more active than compound 9o (Cpd. 83), that had a sulfonyl substituent, and compound 9p (Cpd. 84) with an amino acid unit. Therefore, this suggested that sulfonyl group attachment on the beta-carboline scaffold is responsible for cytotoxic activity. Interestingly, some of these beta-carboline derivatives, especially 9e (Cpd. 73), 9l (Cpd. 80), 9o (Cpd. 83), and 9p (Cpd. 84), were more cytotoxic on the cancer cells than the positive control, 5-fluorouracil    46]..BurkholdAspergillus ochraceus strain LCJ11-102 led to the isolation of four new ochrazepine A, B, C, and D (Cpds. 85–88) conjugates. By the nucleophilic addition to the epoxide, these conjugates dimerized from 2-hydroxycircumdatin C (Cpd. 89) and aspyrone (Cpd. 90) and by the semisynthesis involving a nucleophilic addition of 2-hydroxycircumdatin C (Cpd. 89) to aspyrone (Cpd. 90), Cpds. 85–88 were obtained. Cpd. 85 was cytotoxic to ten human cancer cell lines, Cpds. 86 and 88 were selectively cytotoxic to the U251 cell line, and Cpd. 87 was active against A673, U87, and Hep3B cell lines with IC50 values of 2.5–11.3 µM among the 26 screened human cancer cell lines , as well as the known biogenetically related compound cytosporin D (Cpd. 95) when grown on rice medium. Cpds. 91–94 contain 12, 32-ester linkage of cytosporin D (Cpd. 95), and decipienolide A or B moieties. The new compounds had weak antiproliferative activity against DU145, SW1990, Huh7, and Panc-1 cell lines with the IC50 range of 4.9 to 17.1 µM , and fifteen known Cpds. (97–111). These compounds were tested for anticancer activity, but only diorcinol (Cpd. 97) and versicolorin B (Cpd. 101) had antiproliferative activity against human glioma U87MG and U251 cell lines with IC50 values of 4.4 and 6.2 µM and 11.3 and 30.5 µM, respectively , one meroterpenoid 12-epi-aspertetranone D (Cpd. 113), two drimane derivatives , and five known Cpds. 116, 117, 118, 119, 120 were isolated. These compounds were screened for anticancer activity against the human prostate cancer cell line 22Rv1, human breast cancer cell line MCF-7, and murine neuroblastoma Neuro-2-A cell line. However, only insulicolide A (Cpd. 114) exhibited cytotoxic activity against 22Rv1 and Neuro-2-A cell lines at IC50 values of 3.0 and 4.9 µM, respectively, that are below 20.0 µM (From the Vietnamese marine sediment-derived fungus  20.0 µM  50]..Aspergil121), 20-demethoxy-20-isobutylaminodactyloquinone D (Cpd. 122), and 19-methoxy-dictyoceratin-A (Cpd. 123), and five known related compounds (Cpds. 124–128) from the marine sponge Dactylospongia elegans. These compounds belong to the meroterpenoid class of terpenes. The cytotoxicity assay on the compounds against the human cancer cell lines DU145, SW1990, Huh7, and Panc-1 revealed growth inhibitory activity on the cell lines with IC50 values in the range of 2.33–37.85 µM. Thus, only the compounds with IC50 below 20.0 µM are in Yu et al. isolated three new sesquiterpene quinones/hydroquinones, 20-demethoxy-20-isopentylaminodactyloquinone D , 16-epi-citreohybriddione A (Cpd. 130)) and one known (Cpd. 131) meroterpenoids. Cpd. 129 is a rare meroterpene without the lactone moiety but possesses the cyclopentan-1, 3-dione group. The three compounds were screened for their antiproliferative activity against HepG2, A549, BIU-87, BEL-7402, ECA-109, HelaS3, and Panc-1 cancer cells. Indeed, only Cpd. 129 demonstrated cytotoxic activity with a selective growth inhibition against HepG2 (IC50 = 7.8 µM). However, the study on the mechanism of action of Cpd. 129 showed that it induced apoptosis in HepG2 cells via direct caspase-8-mediated caspase-3 activation. Additionally, a dual luciferase reporter gene assay carried out to investigate the retinoid X receptor alpha (RXRα) transcriptional activity revealed that Cpd. 129 increased the reporter transcriptional activation of RXRα and reduced the trans-activity of RXRα induced by 9-cis-RA ..Spongia 176–A246 (Cpds. 176–246) and eight known (Cpds. 247–254) aphidicolin congeners from a fungus Botryotinia fuckeliana MCCC 3A00494 from the western Pacific Ocean (−5572 m), were reported by Niu et al. The structures of Cpds. 176–246 were determined by spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher’s method, and the ECD exciton chirality method. Cpds. 229–232 and 233–239 are novel 6/6/5/6/5 pentacyclic aphidicolins with tetrahydrofuran and dihydrofuran rings, respectively, and Cpds. 240–246 are rare noraphidicolins. Aphidicolin A183 (Cpd. 183) induced apoptosis in T24 (IC50 = 2.5 µM) and HL-60 (IC50 = 6.1 µM) cancer cells , and kauamide (Cpd. 263), a new polyketide-peptide containing an 11-membered heterocycle. From the spectroscopic analyses, the planar structure of Cpd. 263 was determined; density functional theory (DFT) calculations of the GIAO NMR shielding tensors and advanced Marfey’s analysis of the N-MeLeu residue were used to determine its relative and absolute configurations, respectively. Cpds. 255 and 257 had moderate inhibitory activity against beta-secretase 1 ((BACE1) IC50 > 20 µM), whereas Cpds. 255–263 exhibited mild to potent antiproliferative effects on human glioma (U251) cells  cells (263) did not show any significant biological activity in the two assays , although it was not tested against Panc-1 ..290), an297), a new indole diterpene, together with seven known compounds (Cpds. 298–304), became isolated from the deep-sea fungus Penicillium sp. YPCMAC1. From the structural analysis studies, penicindopene A was the first example of an indole diterpene that possesses a 3-hydroxyl-2-indolone moiety; it was moderately cytotoxic to A549 and HeLa cell lines with IC50 values of 15.2 and 20.5 µM, respectively .424) and a new diketopiperazine (Cpd. 425), in addition to three known compounds (Cpd. 426–428), were isolated from Penicillium crustosum HDN153086, an Antarctic marine-derived fungus. The structures of Cpds. 424–428 were deduced from the MS, NMR, and TD-DFT calculations of specific ECD spectra. The compounds were tested for cytotoxic activities against the K562 cell line. However, only Cpd. 425 (fusaperazine F) is cytotoxic to cancer cells, with an IC50 of 12.7 μM ..Siphonoc463−465) inhibited the growth of P388 murine leukemia cells with their IC50 in the micromolar range - and (S)-configurations that occurred in a 9:91 ratio after the absolute configuration of the substituted anteiso-methyl was assigned ..558–563)Dendronephthya mucronata yielded four bicyclic lactones A–D , which includes three new compounds named dendronephthyones A–C  after a series of chromatographic purifications. From the bioassay screening, Cpds. 565–567 exhibited weak selective cytotoxic activity against the HeLa human malignant tumor cell line with IC50 values of 32.48, 30.12, 35.14, and 14.45 µM (50 > 100 µM) ..644) andAspergillus sp. LS34, a sponge-derived fungus, cultured in two different media (a solid-rice medium and potato dextrose broth (PDB)), two new compounds, asperspin A (Cpd. 648) and asperther A (Cpd. 649), plus seven known Cpds. 650–656, were isolated. The fungal extracts cultured in the rice medium produced Cpds. 648–652 and in the PDA medium, Cpds. 653–656. From the anticancer activity study carried out on the compounds against the seven cancer cell lines, CCRF-CEM, K562, BGC823, AGS, HCT-116, MDA-MB-453, and COR-L23, only Cpd. 656 had significant antiproliferative activity on CCRF-CEM and K562 cells with 1.22 and 10.58 µM IC50 values, respectively (By using the one-strain-many-compound (OSMAC) technique on ectively  107]..AspergilCpd. 657), and eight known triterpene glycosides (Cpds. 658–665), that were obtained from the methanol extract of the Vietnamese sea cucumber Holothuria edulis, were deduced. Holothurin A5 (Cpd. 657) has a hydroperoxy group at C-25. That was the first report of the isolation of this group of triterpene saponin compounds from sea cucumbers ..Cyanobac670), a compound obtained from Aspergillus terreus strain S020, after culturing in a 21 L fermentation static broth for 40 days at room temperature on potato dextrose broth and a series of chemical treatments and chromatographic separations, in a yield of 537.26 ± 23.42 g/kg extract; thus, this represents the highest yield of terrein produced by fermentation to date. After the cytotoxic bioactivity-guided screening of the fungal extracts, the isolated compound terrein was also screened against human colorectal (HCT-116) and hepatocellular (HepG2) carcinoma cell lines. It exhibited cytotoxicity on the cells with IC50 values of 12.13 and 22.53 µM  and prenylcandidusin D-G , were sourced from an endophytic fungus, Aspergillus candidus LDJ-5. Their structures were determined from NMR and MS data. In contrast to a previously reported study on p-terphenyls, Cpd. 676 has a rare 6, 5, 6, 6-fused ring system. With this range (0.40–16.9 µM) of IC50 values, Cpds. 681, 682, 675, and 679 were cytotoxic to a panel of nine human cancer cell lines (673) is in The following nine new prenylated p-terphenyls, prenylterphenyllins F-J , and ten known compounds, four alkaloids (Cpds. 683–686), two isocoumarins (Cpds. 687 and 688), and four polyketides (Cpds. 689–692), were isolated from a marine shellfish-associated fungus Monascus ruber BB5; they were screened for antiproliferative activity against human nasopharyngeal  and hepatocellular (QGY7701 and HepG2) carcinoma cell lines. Monarubin B (Cpd. 680) was potently cytotoxic to HepG2 and QGY7701 with the respective IC50 values of 1.72 and 0.71 μΜ; lunatinin (Cpd. 686) was moderately cytotoxic to HepG2, QGY770, and SUNE1 with the respective IC50 values of 9.60, 7.12, and 28.12 μΜ. ..693) andTrichoderma erinaceum F1-1 in glucose–peptone–yeast (GPY) medium supplemented with a single amino acid, L-phenylalanine, or not. Seven terpenoids (Cpds. 699–705) and one polyketide (Cpd. 706), out of which four are new compounds, namely, harziandione A (Cpd. 699), cyclonerodiols A (Cpd. 701) and B (Cpd. 702), and trichodermaerin A (Cpd. 704), from the GPY medium without L-phenylalanine, and 18 aromatic compounds (Cpds. 707–722), which include six new compounds, named trichoderolides A–F , from the GPY medium with L-phenylalanine were obtained. Cpd. 708, 710, and 711 were cytotoxic to a human melanocyte cancer cell-MDA-MB-435. Cpd. 724 was cytotoxic to A549 human alveolar basal epithelial cell adenocarcinoma , and one known decalin tetramic acid, phomasetin (Cpd. 728), from the C18-SPE fractions of the trichloromethane sub-extract of an endophyte Pyrenochaetopsis sp. FVE-001, a fungus derived from the brown alga Fucus vesiculosus. Cpds. 725 and 726 exhibited antitumor activity against the melanoma neoplasm cell line A-375 with IC50 values of 2.8 and 6.3 µM . Donghaecyclinones A–C (Cpds. 729–731) demonstrated cytotoxic activity against diverse human cancer cell lines (IC50: 6.7–9.6 μM for Cpd. 731)   117]..Streptom50 (≤20.0 µM) values of both known and novel compounds were shown with structures of the new compounds. Interestingly, some of these compounds exhibited a broad spectrum of anticancer activity and are worth further exploration in terms of mechanism of action, structure–activity relationship, and clinical trials. However, this could serve as a guide for the selection of novel compounds/derivatives for future optimization and drug development.There has been a growing rate of cancer incidence worldwide due to factors such as an aging population, eating habits, environmental changes, and co-morbidities. Poor treatment outcomes and complications of cancer disease are aggravated by multi-drug resistant malignant tumors, the high cost of treatment, and adverse drug reactions/patient compliance. Therefore, there is high demand for new anticancer agents or for modifying the existing ones. This necessitated the search for potent and efficacious substances from marine sources that will improve or replace existing ones in order to contain multi-drug-resistant neoplasms. This review compiled a total of 731 compounds/derivatives that belong to these classes: glycosides, alkaloids, saponins, lipids, terpenes, ribose, steroids, peptides, xanthones, ethers, lignins, coumarins, carbazoles, azaphilones, nucleosides, polyketides, and quinones . These c


N-Heterocycles are considered as desirable scaffolds for the development of novel lead compounds for anticancer drug research. Among them, phosphorus-containing amino-derivatives play a crucial role. A series of imines and products of their further reactions with P-nucleophiles were obtained starting from vicinal bisamines. Reaction of ethylenediamine and α-carbonyl esters yielded in novel unexpected products, which structures were confirmed by crystallographic measurements. The cytotoxic activity evaluation was done on a variety of cell lines including HUH7, AKH12, DAOY, UW228-2, D283, D425, and U251. Human umbilical vein endothelial cells (HUVECs) were used as control. Two of the tested compounds, bearing TADDOL-derived, and trifluoromethyl substituents showed a significant effect on cell viability, though comparable to nonmalignant cells. Silica gel 60 was used for both column chromatography and thin-layer chromatography performed with the Merck plates (F254) which were visualized using UV light and iodine vapors.The reactants and solvents were received from commercial suppliers and used without additional purification. All melting points are uncorrected and determined by the open capillary method using ApotecR,6R)-3-oxo-2,5-diazabicyclo[4.4.0]dec-4-ene (1)—dec-4-ene (1′)—dec-4-ene (1″)—,17R,6R)-3-oxo-2,5-diazabicyclo[4.4.0]dec-4-ene (2a)—dec-4-ene (2c)—dec-4-ene (2d)—dec-4-yl]-phosphonic acid—dec-4-yl] phosphonate (4a)—dec-4-yl] phosphonate (4b)—dec-4-yl] phosphonate (4c)—dec-4-yl] phosphonate (4d)—dioxolodioxaphosphepin-6-yl))--3-oxo-2,5-diazabicyclo[4.4.0]decan (4e)—decane (4f)—dec-4-yl]-phosphonic acid (5a)—dec-4-yl]-phosphonic acid (5b)—dec-4-ene (2b). Typical procedure.4-trifluoromethyl--1,2-diaminocyclohexane  was dissolved in 2-PrOH (4 mL). Ethyl 3,3,3-trifluoropyruvate  was introduced to the stirred solution and the mixture was stirred for 24 h at room temperature (293 K). The precipitate was filtered off, washed with three portions of 2-PrOH (3 × 2 mL) and air-dried overnight.. 1H NMR 2DO): δ 7.42 , 3.03 , 2.80–2.86 , 1.84–1.95 , 1.68–1.74 , 1.18–1.42 . 13C NMR 2DO): δ 164.9, 123.4 , 82.7 , 57.3, 53.5, 30.0, 29.8, 24.0, 23.6. IR (KBr): 3351, 3211, 3108, 2949, 2888, 2869, 1679, 1652, 1477, 1427, 1192, 1155, 1061, 1000, 806 cm−1. HRMS : calcd. for C9H12F3N2O ([M + H]+), 221.0902, found, 221.0910.Colorless solid . Mp. 184–185 °C. [α]3,8-Diphenyl-1,4,7,10-tetraazacyclododeca-3,7-diene-2,9-dione (9); Typical procedure2Cl2/MeOH 80:20 v/v).Ethylenediamine  was dissolved in 2-PrOH (20 mL). Ethyl 2-oxo-2-phenylacetate  was then added and the resulting mixture was stirred at 293 K for 48 h. After removal of solvent in vacuo, the products were isolated by chromatography on silica gel column : δ 8.70 , 7.72–7.74 , 7.46–7.52 , 4.08 , 3.40 , 3.03 . 13C NMR : δ 167.3 (2C overlapped), 165.9 (2C overlapped), 135.1 (2C overlapped), 131.3 (2C overlapped), 129.0 (4C overlapped), 127.7 (4C overlapped), 55.6 (2C overlapped), 37.2 (2C overlapped). HRMS : calcd. for C20H21N4O2 ([M + H]+), 349.1664, found, 349.1659. Colorless solid . Mp. 291–293 °C. a]pyrazine-5,10-dione (10). Typical procedure.Hexahydroimidazoimidazo+), 197.1039.Colorless solid . Mp. 185–187 °C. 2 by a full-matrix least squares technique was performed with SHELXL-2014/16 [iso(H) = 1.2Ueq (C) for CH,CH2. All figures were prepared using DIAMOND program [Crystallographic measurements for 8, 9 and 10 were collected at 100(2) K with Κ-geometry diffractometers: Xcalibur Ruby and KM4 Sapphire 2 CCD detector (for 8) equipped with an Oxford Cryosystems cooler, using Mo-Kα radiation . CrysAlisPro software was used for data collection, cell refinement, data reduction and analysis . CrysAli-2014/16  using an program .www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc.cam.ac.uk, or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033.CCDC  contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via 2 atmosphere.The human HCC cell line, HUH7 was purchased from ATCC . The Austrian human HCC cell line AKH12 has been well characterized  and was 4 cells were placed in 24-well plates and grown for 24 h prior to treatment with compounds. After treatment for 24 h, cells were incubated at 37 °C with neutral red dye at 50 µg/mL concentration in serum free medium for 2 h. Cells were washed twice with PBS and incubated with 1% CH3COOH in 70% C2H5OH. Concentration of the dye was determined photometrically at 562 nm. The percentage of survival was expressed as ratio of survival against cells treated with vehicle . TD50 was defined as the concentration of compound required to eliminate half of cells after a specified test duration. Neutral red assay was applied for cell viability determination as described previously . 3 × 1044 D425Med cells were seeded in 24-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO2. The cells were treated with investigated piperazin-2-one derivatives for 24 h. A cell viability assay was performed using the CellTiter Blue Reagent  in agreement with the manufacturer’s protocol. As the method for the cell viability was changed for this cell line, we applied the same method for HUVEC cell line for comparison. Three independent experiments were performed. The percentage of survival was expressed as ratio of survival against of DMSO-treated cells (indicated as 0 µM concentration).One day before treatment 2 × 10The release of lactate dehydrogenase into culture media was determined by a cytotoxicity detection kit from Roche Diagnostics according to the manufacturer’s instruction. Triton X 100-lysed cells with the maximum LDH activity were considered as a 100% positive control. DMSO used as the vehicle control.R,6R)-1 was prepared from -1,2-diaminocyclohexane (DACH) and ethyl glyoxylate as described previously [1 was also prepared as  enantiomer (1′) as well as a racemate (1″). A series of enantiopure 4-substituted derivatives 2a–2d were obtained from DACH and ethyl esters of ketoacids [Chiral, enantiomerically pure imine (1eviously  (Scheme etoacids .H)-one 3 was prepared from o-phenylenediamine and ethyl glyoxylate  or exclusively one diastereomer (for 5b).In addition, two phosphonic acids 5a  and 5b [5a [5b  were pre6, by conducting the reactions of ketoesters with ethylenediamine. The results of these preparations were found dependent on the structure of carbonyl substrate. The presence of highly electrophilic –CF3 moiety led to the formation of hydrate form of imine in a quantitative yield piperazin-2-one ve yield . 7, a C2-symmetrical amide 8 and a macrocyclic compound 9 were isolated (Reaction of ethylenediamine and ethyl benzoylformate yielded a complex mixture from which three main products: a cyclic imine isolated . The strisolated . 6–9, a tricyclic dilactam 10 was formed. This unexpected product can pose as an useful scaffold for organic synthesis. The structure of compound 10 in chloroform solution was established by 2D NMR techniques, and was identical with solid state structure obtained from X-ray measurements (To our surprise, ethylenediamine reacted with ethyl glyoxylate in a completely different manner: instead of analogs of compounds urements . It is aN-tosyl-ethylenediamine with glyoxal in methanol [N-heterocycle in the presence of transition metal salts as catalysts. Various molar ratio directed the reaction towards synthesis of monocyclic imine, tricyclic tetraamine or double bicyclic structure. Our approach is quite simpler, and the use of catalysts was not necessary. We observed a dependence between ethyl glyoxylate form used and the yield of tricyclic product. As seen in 10 with a relatively good efficiency. A similar aza-heterocyclic scaffold was obtained by Li and co-workers via a five-component cyclization of methanol . The aut9 and 10 were excluded from the cellular in vitro assays.Using a presented set of reactions, we obtained a series of various nitrogen-based heterocycles for cytotoxic activity tests. Structural variations of the compounds was applied to broadly evaluate and discuss the structure-activity relationship. Due to the poor solubility in DMSO, compounds The general approach to the evaluation of biological activity of investigated compounds followed the one described in our previous study on sulfonamides based on 2-azanorbornane skeleton . All com4e and 6 reduced cell viability below TD50 in HUH7 and AKH12 cell lines (HCC accounts for a vast majority of liver cancer and is among the leading causes of cancer-related mortality with the highest incidence in Northern and Western Africa and Eastern and South-Eastern Asia . The intll lines . The cytll lines . The effll lines .4e and 6 with 50 µM concentration reduced cell viability in all 4 cell lines (50 values for the investigated compounds in all MB cell lines and HUVEC cells are shown in MB considered being the most common malignant brain tumor, comprises a biologically heterogeneous group of embryonal tumor with an estimated 5000–8000 cases per year worldwide . In currll lines . LDH rel4e and 6 decreased cell viability by more than 50% (50 values of tested compounds on U251 and HUVEC cell lines.Despite aggressive therapy glioblastoma is still associated with a dismal prognosis in almost all adult patients . U251 glthan 50% . The LDHthan 50% . Howeverthan 50% . Table 21, its enantiomer 1′, and their equimolar mixture 1″ Interestingly, for two cancer lines racemate exhibited the highest activity among these samples, although the effect was still unsatisfactory. Significantly better results were obtained for two derivatives: a phosphonate bearing big TADDOL—derived moiety 4e, and a monocyclic diamine with trifluoromethyl substituent 6. Compound 4e was previously tested in our previous antiproliferative study using other cancer lines and was found as the only active compound [The results of our study show that most of the investigated compounds have a negligible effect on the cell viability of the lines tested. A weak activity can be noticed for compound compound . Moreovecompound . Apparen6. This piperazine-based compound differs from the rest of the investigated compounds that bear a bicyclic skeleton. While NH, OH and C=O fragments offer numerous possibilities of interactions with biological targets by hydrogen bonds, fluorinated groups, and, in particular, CF3 substituent are well recognized pharmacophores [A similar activity in the current study was observed for a lactam containing trifluoromethyl group cophores ,41,42. U10. Another product bearing CF3 substituent exhibited a significant cytotoxic activity. Reaction of cyclohexanediamine with ketoesters serves as a useful route to chiral cyclic imines which can be converted to a variety of derivatives, including aminophosphonates. Here, checked also the possible use of other diamines in a similar transformation, and found that in case of ethylenediamine the course of the reaction is strongly dependent on the structure of the second reactant. This led to preparation of interesting amides, including a chiral bicyclic dilactam 4e and 6 decrease cell viability by more than 50% in all cell lines tested. Unfortunately, similar compound toxicity was observed in nonmalignant HUVEC cells. Further efforts to improve compound selectivity towards cancer cells are required. We examined the effect of the prepared compounds on a variety of malignant cell lines, including HCC, MB and GBM. HUVEC, a non-malignant primary cells were used as a control. Our results indicate that 50 µM concentration of compounds


Parp-1−/− mice show higher susceptibility to the carcinogenic effects of nitrosamine and azoxymethane. To elucidate the role of alterations of the PARP-1 gene in human carcinogenesis, we examined the expression level of PARP-1 gene in various human tumor cell lines. The presence of gross rearrangement of PARP-1 gene in these cell lines was also examined by Southern blot hybridization analysis. The expression levels of PARP-1 gene in several cell lines, including T-cell leukemia cell lines (Molt-4 and CCRF-CEM), colon cancer cell line (WiDr), and gastric cancer cell lines  was substantially lower than in other cancer cell lines. Among the 85 analyzed cell lines, structural alteration of PARP-1 gene was detected in a gastric cancer cell line, MKN28. A low level of PARP-1 expression in human cancer could potentially influence cancer cell growth, differentiation and cancer development by affecting genomic instability, as well as the response of tumors to chemo- and radiotherapy.Poly(ADP-ribose) polymerase-1 (Parp-1) is involved in DNA repair and cell-death induction after DNA damage.  Parp-1−/− mice, a higher frequency of deletion mutations was observed than in Parp-1+/+ mice after treatment with carcinogenic N-nitrosobis(2-hydroxypropyl)amine (BHP),Parp-1 deficiency.8)Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes polyADP-ribosylation of proteins using NAD as a substrate after activation by DNA strand breaks.PARP-1 gene in human cancer has not been fully studied yet, but several reports described the level of PARP-1 gene expressionPARP-1 gene in various malignancies.et al.PARP-1 on chromosome 13q33-qter is increased in Burkitt lymphoma, multiple myeloma, colon and prostate cancers in African American populations. Furthermore, Bieche et al.PARP-1 gene expression correlated with higher genomic instability in breast cancer. Prasad et al.PARP-1 mRNA expression.Although the evidence suggests that PARP-1 is likely to be involved in human carcinogenesis, alteration of the Parp-1−/− cells show enhanced sensitivity to alkylating agents and γ-irradiation,PARP-1 mRNA in various tumor cell lines and the presence of gross structural alterations in the PARP-1 gene by Southern blot hybridization analysis.Since Studies were conducted using 11 colon cancer cell lines , SW1116, LMCO5, Colo201, Colo205, LMCo3, LMCO4, PMCO1, Colo320DMF, and Colo321), 8 liver cancer cell lines , 9 gastric cancer cell lines ,22)PARP-1, encompassing the entire coding region of the PARP-1 gene.23)Northern and Southern blot analyses were performed as described previously.32P-NAD  into an acid-insoluble fraction as described previously.2, 1 mM dithiothreitol, 2 μg/ml activated DNA , 2 μg/ml calf thymus histone (Sigma), and 100 μM 32P-NAD (8 Ci/mmol) at 25 °C for 10 min. The reaction was stopped by addition of trichloroacetic acid to 5% and the radioactivity in precipitate was measured after filtration through glass-filter . One unit of PARP activity was defined as an incorporation of 1 pmol of NAD per min at 25 °C.PARP-1 activity was measured by incorporation of radioactivity derived from PARP-1 mRNA expression was analyzed by northern blot analysis in 9 colon tumor cell lines, 7 liver cancer cell lines, 6 leukemia cell lines, and 4 sarcoma cell lines . Previous studies reported the association of higher B allele frequency with endemic Burkitt lymphoma, multiple myeloma, colon and prostate cancers in African American populations.et al.ll lines . Colon cll lines ,26 and all lines  showed lomic DNA , arrow. PARP-1 gene compared to six other gastric cancer cell lines . These results suggest that MKN28 contains the structural alteration in the PARP-1 gene. When the PARP-1 cDNA probe, encompassing the entire coding region, was divided into N-terminal, automodification, and C-terminal domains, the extra fragment “a” hybridized only to the C-terminal probe, whereas the fragment “b” hybridized to both automodification and C-terminal probes. This suggests that the structural alteration spans the auto-modification to C-terminal domains of the PARP-1 gene. The probe used for Southern blot hybridization analysis is a cDNA encompassing the whole coding region of the PARP-1 gene and it detects PARP-1 gene on chromosome 1q41-q42 and pseudogenes on chromosome 13q33-qter and on chromosome 14q22.Furthermore, MKN28PARP-1 mRNA expression level implies that the structural alteration in MNK28 did not affect the basal activity of PARP-1. It is yet to be clarified whether the enzymatic activity or the function of PARP-1 under DNA damaging conditions is influenced by this structural alteration of the PARP-1 gene in MKN28.We measured PARP-1 activity using cell extracts of MKN28 and MKN45, as shown in PARP-1 gene was observed in these samples by Southern blot hybridization analysis with EcoRI digestion indicating that gross rearrangement in the PARP-1 gene may be a rare event in cancer cells.Gene rearrangement was further screened in 48 lung cancer cell lines , 13 esophageal cancer cell lines , 3 cervical cancer cell lines , 3 colon cancer cell lines , 5 renal cancer cell lines  and 13 lymphoma/leukemia cell lines , and 3 primary lung cancer tissues (data not shown). No rearrangement in the PARP-1 gene mutation and polymorphism should be conducted at the sequence level by refined methods, including PCR-SSCP (single-strand conformation polymorphism) and direct sequencing. Reduced polyADP-ribosylation activity was demonstrated in patients with Werner Syndrome after treatment with an alkylating agent, methylmethanesulfonate, but was not observed without any treatment or after treatment with bleomycin,To determine the role of PARP-1 dysfunction in carcinogenesis, further analysis of PARP-1 gene. A low level of PARP-1 expression in human cancer could have a substantial impact on cancer cell growth, differentiation and cancer development by affecting genomic instability, as well as on the response of tumors to chemo- and radiotherapy.In conclusion, we identified in this study those cancer cell lines with low level of expression and structural alteration of


Breast cancer is one of the major causes of mortalities noticed in women globally. DDX3 has emerged as a potent target for several cancers, including breast cancer to which currently there are no reported or approved drugs.in silico molecular docking studies have shown that the compounds have occupied the binding site of the human DDX3 target. Furthermore, to investigate the cell viability effect of 3a, 3b, and 3c on MCF-7 and MDA-MB-231 cell lines, the cell lines were treated with different concentrations of compounds for 24 and 48 h and measured using MTT assay.To find effective cancer therapeutics, three compounds were computationally designed tweaking the structure of natural compound butein. These compounds were synthesized and evaluated for their anticancer property in MCF-7 and MDA-MB-231 cell lines targeting DDX3. The 3b and 3c have reduced the expression of DDX3 in MCF-7 and MDA-MD-231 cell lines. 3b or 3c treated cell lines increased apoptotic protein expression. Both the compounds have induced the apoptotic cell death by elevated levels of cleaved PARP and cleaved caspase 3 and repression of the anti-apoptosis protein BCL-xL. Additionally, they have demonstrated the G2/M phase cell cycle arrest in both the cell lines. Additionally, 3c decreased PI3K and AKT levels.The cell viability results showed that the have induced dose dependent suppression of DDX3 expression. Additionally, Our results shed light on the anticancer ability of the designed compounds. These compounds can be employed as chemical spaces to design new prospective drug candidates. Additionally, our computational method can be adapted to design new chemical scaffolds as plausible inhibitors. RNA helicases are a group of proteins possessing a unique motif, called the DEAD/H (Asp-Glu-Ala-Asp/His) . One of via the RNA and demonstrates a double role in viral replication. Intriguingly, it works as a cofactor for viral replication and acts as a mediator of the innate immunity system . The compound was minimized by employing the Full Minimization protocol accessible with DS.The butein structure was downloaded from the PubChem database  in the 2Discovery Studio, the B ring of butein was modified to obtain new structures as described in Using the Random Ligand Conformations are generated using high-temperature MD. These conformations are then translated into the binding site. The candidate poses are subsequently created using random rigid-body rotations followed by simulated annealing. A final minimization is then used to refine the ligand poses  for 1H NMR spectra are given in ppm relative to TMS. Column chromatography was performed on Merck silica gel (40–63 mesh).Unless otherwise noted, all reactions were carried out under an atmosphere of argon in oven-dried flasks. All reagents and chemicals were purchased from Sigma Aldrich Co., TCI, or Alfa Aesar Co. Solvents, such as tetrahydrofuran (THF) and methylene chloride, were used after distillation following standard purification procedures. 2Cl2 (5 mL) triethylamine  and 4-dimethylamino pyridine  were added, and then tertbutyldimethylsilyl chloride  dissolved in CH2Cl2 (9 mL) was added dropwise. After the completion of addition, the reaction mixture was allowed to stirred overnight at room temperature. The reaction mixture was quenched by the addition of water. The organic fractions were collected, and the aqueous phase was extracted with CH2Cl2. The combined organic fractions were washed with water and brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by short column chromatography on silica gel using n-hexanes as the eluent to afford compound 2  as a colorless oil; 1H NMR  δ 7.62 , 6.47 , 6.32 , 2.57 , 1.00 , 0.97 , 0.28 , 0.21 .To a solution of 2′,4′-dihydroxyacetophenone  in dry CH2 (1.0 mmol) in dry THF (3 ml) under argon was added lithium diisopropylamide  dropwise at −78°C. The reaction mixture was allowed to warm to −20°C over 30 min, and then cooled to −78°C. The solution of substituted aldehyde (1.0 mmol) in dry THF (3 ml) was added dropwise at −78°C, and the resulting reaction mixture was allowed to warm to room temperature stirred overnight and then quenched by addition of sat. aqueous NH4Cl. The organic fraction was collected, and the aqueous layer was extracted with diethyl ether. The combined organic fractions were washed with brine, dried over anhydrous MgSO4, and evaporated under reduced pressure to afford the monohydroxy protected chalcones. The monohydroxy protected chalcones were purified by using CH2Cl2/MeOH as eluent.To a solution of compound 4, and evaporated under reduced pressure. The residues were purified column chromatography on silica gel using CH2Cl2/MeOH as eluent to afford compounds 3a-3c in 70% to 85% yields.The monohydroxy protected chalcone derivatives (0.2 mmol) were dissolved in THF (5 ml), and tetrabutylammonium fluoride  was added. The resulting reaction mixture was stirred at room temperature for 30 min. and quenched by addition of water. The organic fraction was collected, and the aqueous layer was extracted with ethyl acetate three times. The combined organic fractions were washed with brine, dried over anhydrous MgSO 3a) was synthesized by following the general procedure using monohydroxy-protected chalcone  dissolved in THF (8 mL) and tetrabutylammonium fluoride . The 3a was purified using silica gel column CH2Cl2/MeOH 10: 1, v/v. Rf. Value = 0.5. Yellow solid ; 1H NMR  δ 13.28 , 7.87 , 7.75 , 7.23 , 7.12 , 7.07 , 6.46–6.40 , 5.53 .The compound ( 3b) was synthesized by following general procedure using monohydroxy-protected chalcone  dissolved in THF (8 mL) and tetrabutylammonium fluoride . The 3b was purified using silica gel column CH2Cl2/MeOH 10: 1, v/v. Rf. Value =0.5. Yellow solid ; 1H NMR  δ 13.34 , 7.84 , 7.59–7.38 , 6.67 , 6.48–6.40 , 5.30 .The compound ( (3c) was synthesized by following general procedure using monohydroxy protected chalcone , was dissolved in THF (2 mL) and tetrabutylammonium fluoride . The 3c was purified using silica gel column CH2Cl2/MeOH 10: 1, v/v. Rf. Value = 0.5. Yellow solid   δ 13.36 , 7.90–7.83 , 7.56 , 7.46 , 7.23–7.35 , 6.47–6.41 , 5.47 , 2.41 ; CAS. No. 1385668-91-6.The compound mg, 77%) ; 1H NMR Human MCF-7 and MDA-MB-231 cell lines were purchased from the Korean Cell Line Bank . The MCF-7 and MDA-MB-231 cell lines were maintained in RPMI-1640 medium  containing 10% (v/v) fetal bovine serum  and 1% penicillin–streptomycin (Gibco) at 37°C in a humidified atmosphere of 5% CO2 .4 cells in 500 µL medium per well for 16 h. The cultured cells were treated with various concentration for 24, 48, and 72 h. After incubation, the cells were added with 55 µl of 5 mg/ml 3--2, 5-diphenyltetrazolium bromide  solution for 2 h, and the medium was removed, which was followed by lysis with DMSO. The absorbance at 570 nm was measured with PowerWave HT microplate spectrophotometer .The cell viability assay was conducted as described before  the cellThe Western blot analysis was performed as described before . After 4The data were expressed as mean ± standard error of mean (SEM) and analyzed by GraphPad Prism Version 4.0b , for statistical significance using one-way analysis of variance (ANOVA). p < 0.05 was considered as statistically significant. All experiments were performed in triplicates.The prepared butein compound was used as the starting structure (parent structure) to computationally design new compounds. Subsequently, modifying the ring B has yielded three compounds as shown in The synthesized compounds were assessed for the binding affinity studies using the CDOCKER program. These compounds were docked into the ATP binding pocket of the DDX3 to understand their potentiality along with butein for comparative study. The results have shown that the modified compounds have demonstrated a better to comparable dock score than the parent compound butein as shown in 3a has prompted hydrogen bond interactions with the residues, Thr201 and Gln207, as shown in 3b has interacted via hydrogen bonds with the residues, Thr231, Ala232, Gln281, and Glu285, respectively, as represented in 3c has formed hydrogen bond interactions with the residues, Thr231, Ala232, and Gln281 residues. The residue Thr231 has formed two hydrogen bonds with the ligand as illustrated in Further, the compounds have shown interactions with the key residues positioning at the binding pocket as elucidated in 1H NMR spectrum data of the three compounds is provided as, 3a, 3b, and 3c, respectively.The syntheses of chalcones are illustrated in 3b and 3c, MTT assays were carried out on the MDA-MB-231 and MCF-7 representing the human breast cancer cell lines. 3b and 3c were treated with various concentration for 24, 48, and 72 h in the MDA-MB-231 and MCF-7, respectively. The cell viability on the MDA-MB-231 and MCF-7 cells was markedly decreased by 3b and 3c treatment in a dose- and time-dependent manner as shown in 50 values of 3b were recorded as 58.23 μM and 37.74 μM at 48 h in MCF-7 and MDA-MB-231 cell line, respectively. Also, IC50 values of 3c were shown as 22.72 μM and 20.51 μM at 48 h in MCF-7 and MDA-MB-231 cell line, respectively. These results showed that 3b and 3c have anti-proliferative effect in both human cell lines. Therefore, we used 20 and 30 μM of 3a, 25 and 50 μM of 3b, and 10 and 20 μM of 3c in our subsequent experiments.To evaluate the anti-proliferative effect of 3a, 3b, or 3c, respectively. The DDX3 protein expression was decreased dose-dependently in 3b or 3c treated MCF-7 and MDA-MB-231 cell lines as shown in 3b and 3c could suppress DDX3 protein expression in MCF-7 and MDA-MB-231 cell lines. Since 3a did not show any significant reduction of DDX3 expression, as shown in Based on cell viability results, we also identified the DDX3 protein expression. The two cell lines, which include MCF-7 and MDA-MB-231 cells were treated with indicated concentration of 3b and 3c in human breast cancer cell lines, we identified the apoptosis and cell cycle related protein expression in 3b and 3c treated MCF-7 and MDA-MB-231 cell lines. The apoptosis marker proteins, such as cleaved PARP and cleaved caspase 3 were increased, and anti-apoptosis protein, BCL-xL, was decreased in compound use to treat both cell lines, as shown 3b and 3c can induce apoptotic cell death and G2/M phase cell cycle arrest in MCF-7 and MDA-MB-231 cell lines, indicating anti-cancer effect, as shown in To determine the cell death and cell cycle arrest effect on 3b and 3c treated human breast cancer cells. 3b was not affective to PI3K/AKT protein change. The results showed that inhibition of PI3K and AKT activation was induced in the 3c treated both cell lines, as shown in In addition, PI3K/AKT signaling pathway is an important intercellular signal involving apoptotic cell death and cell cycle arrest. Therefore, we examined the phosphorylated PI3K and AKT protein expression in the 3a, 3b, and 3c, were docked into the active site of the protein target 2I4I to gain insights into the atomistic interactions. Alluringly, the three compounds have rendered an interaction with the key residue Tyr200, via π-π stalk interaction with 3a and 3c, whereas 3b has prompted a π-alkyl interaction as shown in With an objective to find effective therapeutics to breast cancer, the current investigation has proceeded by tweaking the natural compound butein. Correspondingly, we have obtained three compounds. These three compounds, namely in vitro MTT assay. This assay has shown a remarkable decline in the cell viability when compared with the untreated cells. The 3a has shown an IC50 value of 27.44 and 26.06 μM in MCF-7 and MDA-MD-231 cell lines for 48 h, whereas the 3b and 3c have demonstrated 58.23 μM and 37.74 μM and 22.72 μM and 20.51 μM, respectively, in MCF-7 and MDA-MD-231 cell lines recorded at 48 hrs. Although, 3a showed relatively stronger IC50 value, it has not repressed the DDX3 expression. This gives rise to the notion that 3a might act on other target other than DDX3, while the 3b and 3c have decreased the expression of DDX3 in both the cell lines. This finding sheds light on the identification of new inhibitors against DDX3.To elucidate on their anti-proliferative activity in cancer cells, we proceeded with the Fundamentally, the flavonoids promote the anticancer mechanism by instigating the apoptosis and cell cycle arrest . The apoThe antiapoptosis protein (apoptosis inhibitor) is an eminent member of BCL-2 family of apoptosis regulators . This pr3b and 3c on cell cycle. Cell cycle has four phases that transvers from quiescence (G0 phase) to proliferation . The process of cell cycle occurs by the activation of cyclin-dependent kinase (CDK) and its corresponding cofactor the cyclins /protein kinase B (AKT) signaling pathway is believed to be associated with the modulation of several cellular physiological processes  and has In conclusion, the present manuscript predominantly focuses on finding new therapeutics targeting DDX3. DDX3 is widely noted target that is over expressed in several cancers. In here, the phytocompound butein has been modified at ring B position to obtain new derivatives. These new compounds have repressed DDX3 expression in two cell lines, illuminating their potential to target DDX3. Additionally, these compounds have arrested the cell cycle proteins and have shown apoptosis, remarkably extending their usability in cancer treatments. Taken together, we propose two butein derivatives as novel DDX3 inhibitors that can additionally serve as scaffolds for designing new compounds.The original contributions presented in the study are included in the article/SR, GL, MK, and KL conceived the idea. SR and GL performed the computational designing and molecular docking. BS and JK have synthesized the compounds. SK and GK have performed the MTT assay and the western blot analysis. SR, BS, and SK wrote the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) and funded by the Korean government (MSIT) (No. NRF-2018M3A9A7057263). This research was supported by the Neurological Disorder Research Program of the National Research Foundation (NRF) funded by the Korean Government (MSIT) (2020M3E5D9080660).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.


Figure 4 as published. Figure 4E depicts the effect of pre-incubating liposomes with either ZIKV or cells, but the legend mistakenly says SEV rather than liposomes. The correct legend appears below.In the original article, there was a mistake in the legend for Figure 4. Lipid content of SEV blocks ZIKV infection. (A) The protein content of SEV was denatured by heating at 95° for 8 min. Nanoparticle tracking analysis (NTA) profile comparing heat-treated and untreated SEV showing that the size profiles do not change. Size profiles of liposomes (see part C) are also plotted. (B) Equal amounts of heat-treated or untreated SEV were incubated with ZIKV virions at a 106 SEV:PFU, then added to epithelial cell lines for 1.5 h before washing and assessing ZIKV binding by ddPCR. As a control, ZIKV virions were also heated at 95° for 8 min. Each line is a separate cell line, points are mean of two replicate wells and error bars are standard deviation. Significance by one-way ANOVA with Tukey's multiple comparisons test. **p > 0.01. (C) ZIKV was pre-incubated with 106 SEV or 106 liposomes for 1 h prior to infecting genital epithelial cells. Seventy-two hours post-infection cells were lysed and ZIKV genomes quantified by ddPCR. Each line is the average of duplicate wells from a separate experiment. Reduction in ZIKV genomes was significant by one-way ANOVA with Dunnett's multiple comparisons test. *p < 0.05, and **p < 0.005. (D) The data from C, and additional conditions with 105 SEV or liposomes, are plotted as percent reduction from ZIKV alone. Each symbol represents the average of two technical wells from a different cell line. The horizontal lines are the mean for each condition. (E) Comparison of pre-incubation of either ZIKV or epithelial cells with liposomes on viral infection. Liposomes were pre-incubated with ZIKV (ratio:106 liposome per PFU) or with cells (same amount of liposomes) for 1 h at 37°, then virus was added to cells for 1.5 h. Cells were washed and cell-associated ZIKV genomes quantified by ddPCR. Each symbol represents the average of two technical replicates from different epithelial cell lines. Significance by one-way ANOVA with Dunnett's multiple comparisons test. *p < 0.05.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


E. coli) and Gram-positive Staphylococcus aureus (S. aureus), but CDFs exhibited better antibacterial performance, and S. aureus could be completely inhibited with the minimum inhibitory concentration of 30 μg/mL. This reveals CDFs magnify both the stability and antibacterial activity, which would be more promising for practical applications.Carbon dots (CDs) have been widely used as antimicrobials due to their active surface, but some CDs suffer instability. Therefore, the relative applications such as the antibacterial activity may not be reliable for long-term use. Herein, we synthesize CDs with blue fluorescence by a hydrothermal process. Thereafter, polyethylenimine was applied for the assembly of CDs into CDs-based frameworks (CDFs). The CDFs exhibited quenched fluorescence but showed more stable properties based on the scanning electron microscope and zeta potential investigations. Both CDs and CDFs show antibacterial activity toward Gram-negative Escherichia coli (The online version contains supplementary material available at 10.1186/s11671-021-03582-3. Bacterial infections show a serious threat to human lives, and the development of effective medicines to disinfect bacteria is in great demand . VariousE. coli) and Gram-positive Staphylococcus aureus (S. aureus) compared to CDs, indicating their broad-spectrum antibacterial performance, though many CDs only eradicated Gram-positive bacteria [To meet the demand for practical applications, the antimicrobials should have the following characteristics: (a) Excellent stability remains unchanged for a certain time in an ambient environment; (b) Excellent biocompatibility and low toxicity: (c) high antibacterial activity. The larger nanomaterials tend to be more stable, but they might have relatively weaker antibacterial activity due to the smaller active surface area. Considering both the weakness of small and large nanomaterials, we report the assembly of the small CDs into large CDFs by simply adding polyethyleneimine (PEI) Fig. . CDs werbacteria . In addibacteria . This woX-ray surface photoelectron spectra (XPS) were recorded on an ESCALAB250Xi X-ray surface photoelectron spectroscopy (XPS) instrument. The transmission electron microscope (TEM) was performed by a JEM-2100 microscope operating at 200 kV. The fluorescence of the materials was obtained using the F97 fluorescence spectrometer. The FDA/PI staining of the bacterial cells was recorded in tapping mode with a Leica DFC450C microscope. Fluorescence lifetime was measured on a time-correlated single-photon counting (TCSPC) system using a Nanolog spectrofluorometer . Ultraviolet–visible spectroscopy (UV–vis) spectra are obtained from the UV-1600 instrument. The bacterial imaging was observed by a confocal microscope (Olympus FLUOVIEW FV1000 c). All the reagents were of analytical grades. The deionized water was used through the experiments. Cell counting kit-8 was obtained from Beyotime Biotechnology.L- cysteine (1.0 g) was dissolved in 10.0 mL of deionized water and mixed well. Then, the pH of the solution was adjusted to 9.0 with 1.0 M NaOH. The solution was transferred to a hydrothermal reactor and heated at 160 °C for 24 h. After the solution was cooled to room temperature, the resulting solution was subjected to dialysis using a dialysis bag (MW 7000 cut off) for one day. The as-obtained CDs were used for the following characterizations and experiments. For the preparation of CDFs, 40 µL of 1% PEI was added to 1 mL of the CDs. The mixture was allowed to stay for 1 h. The product was purified by dialysis using the same method as the purification of CDs. For HR-TEM characterization, the samples were concentrated into a small volume, transferred to a silica gel column, and eluted with methanol and dichloromethane to obtain the further purified products.PC12 cells were seeded in 96-well plates for 12 h and then were incubated with CDs and CDFs with different concentrations. The number of viable cells was investigated using a Cell Counting Kit-8 assay (CCK-8). 3--2, 5-diphenyltetrazolium bromide (MTT) (5 mg/mL in PBS) was added at 1/10 culture volume, and the cells were returned to the incubator. After that the supernatants were discarded and 200 μL of dimethyl sulfoxide (DMSO) was added to each well. The crystals were dissolved by shaking the plates for 10 min. The absorbance at 490 nm was measured using the Microreader (Varioskan LUX Multimode Reader). Blank control wells were included for all the absorbance measurements.E. coli and S. aureus were incubated in the absence and presence of CDs and CDFs at 37 °C with 250 rpm shake. Growth of the bacterial cells in Lysogeny broth (LB) culture was measured by the Microreader at 600 nm wavelength (OD600). LB medium was used as the blank control. The OD600 stands for the cell density, and the relative cell viability was calculated based on the comparison between the cultured bacterial cells in the presence of the materials to the control group . The live/dead bacteria are evaluated by the FDA/PI staining protocol [protocol .Intracellular ROS production was measured in bacterial cells before and after the treatment by CDs and CDFs for 12 h, with a 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe following the instructions. The fluorescence was detected at an emission wavelength of 525 nm with excitation at 488 nm.Figure Zeta-potential investigations were used to measure the degree of electrostatic repulsion of CDs and CDFs between adjacent charged particles in the dispersed system Fig. . It can The fluorescence emission spectra were used to monitor the assembly process of CDs to CDFs Fig. . As the For evaluation of the safety of the materials, the toxicity of the CDs and CDFs to PC12 cells is investigated. Assays of MTT were conducted to investigate the influence of the materials on cell viabilities Fig. . After iS. aureus and E. coli cells. Especially, the viability of the S. aureus cells was almost 0 when larger than 30 µg/mL of the CDFs were used. Similarly, the E. coli cells were disinfected by both CDs and CDFs. CDs showed multiple charges ,HR-TEM (b), and AFM  of the as-obtained CDs; Figure S2: SEM of CDscharacterized at four random times; Figure S3: (a) UV-vis, (b) the fluorescenceexcitation and emission spectra of the CDs; Figure S4: XPS survey (a) and FTIR ofthe as-obtained CDs; Table S1: Lifetime of the CDs and CDFs.


Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established. Based on levels of the estrogen, progesterone and HER2 receptors, breast cancer can be divided in different subtypes. The triple-negative subtype (TNBC) lacking the expression of these three hormone receptors accounts for 15–20% of all tumors2 and is the most aggressive subtype, often leading to metastases4. Despite the efforts, there is still no targeted therapy for TNBC available5. A major reason for this lack in clinical translation may be the use of two-dimensional in vitro experiments that do poorly represent the three-dimensional (3D) tissue physiology observed in human cancer patients. To increase translation from in vitro findings to a clinical setting, different 3D culture systems are now explored, such as organoid cultures, patient-derived xenograft models, reprogrammed stem cell like models, tumor-on-a-chip and 3D cultures of immortalized breast cancer cell lines6. While the majority of breast cancer drug screening studies in the last decade have still been performed in 2D12, there is an increasing number of drug screens performed in more complex models such as patient-derived organoids14, tumor-on-a-chip15 and patient-derived xenograft16 models. Although these complex models better represent human physiology and should increase clinical translation19, drawbacks of these models include reduced reproducibility21, increasing costs, inconvenient maintenance, difficulties in expanding them and generating genetic modifications, making these models less suitable for high-throughput screening22. Next to the already widely studied phenotypic changes between different culturing models23 and phenotypic classification of different tumor subtypes6, transcriptomic and proteomic analyses can contribute to the understanding of the differences between established in vitro models and help to determine the most suitable model in terms of both clinical translation, costs and efficiency.Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women with an estimated 40,610 deaths in the United States in 2017Here, we performed RNA-sequencing of 14 breast cancer cell lines cultured on a 2D plastic substrate as well as in a 3D matrigel-collagen environment. In this 3D model, cells spontaneously form spheroid-like structures exhibiting cell–cell as well as cell–extracellular matrix interactions, thereby changing their cell polarity and shape. We unraveled the transcriptomic differences linked to the invasive phenotype of basal B (or claudin-low) TNBC compared to basal A and luminal breast cancer and uncovered a spectrum of genes higher expressed in 2D cultures that were related to metastatic progression in breast cancer patients. Since the transcriptomic correlation of in vitro cultured cell models to patient tumor tissue was highly subtype and pathway dependent, we established a bioinformatics tool that can be used in future studies to select the most suitable cell type and culture conditions for the pathway of interest. Altogether, this study unraveled the transcriptomic variance between different breast cancer in vitro models and provides an important database that can contribute to selection of the most effective and relevant drug candidates for the treatment of TNBC.28 with selected cell lines representing the different BC subtypes ). These cell line subtypes were validated in our RNA sequencing dataset; hierarchical clustering based on RNA expression of previously published luminal and basal markers clearly separated the different subtypes  cells, we performed RNA sequencing of 52 human breast cancer cell lines cultured on 2D tissue culture plastic and 14 cell lines cultured in a 3D matrigel-collagen environment   tumors) derived from The Cancer Genome Atlas (TCGA). Since we observed clear differences in culture-specific gene expression changes for the different subtypes, we performed these analyses for the DEGs obtained from the paired analysis for all cell lines, basal A and basal B specific DEGs Fig. A-B. Sinc42. The respective targets of these various inhibitors were significantly lower expressed in 3D cultures  and showed that the sensitivity to EGFR inhibitors such as gefitinib is determined by the variation rather than the mean EGFR expression; PDX models bearing heterogeneous expression of EGFR were more sensitive towards gefitinib46. EGFR-high subpopulations were characterized by growth inhibition upon gefitinib treatment and enhanced expression of mesenchymal-stem cell markers46. Interestingly, the same mesenchymal-stem cell markers were also more highly expressed in non-invasive EGFR-inhibitor sensitive cells in both 2D  uncover the differential pathway expression linked to subtype-specific morphological changes and (2) identify the most suitable model for future drug screens. Consistent with previous comparisons of 2D and 3D cultures28. Also needs to be noted that the MDA-MB-231 cell line that was classified as basal B and has been used in this context in many studies, demonstrated to be a poor basal cell line representative in a recent study48. These contrasting phenotypes between basal A and basal B cells are likely related to differences in expression of a small subset of extracellular matrix interacting and cell–cell adhesion genes. Thus, the basal B cells upregulated genes involved in collagen biosynthesis and breakdown of the extracellular matrix in 3D cultures, while these genes were downregulated in basal A cells. Moreover, the higher basal levels of E-cadherin in the basal A subtype could contribute to the less invasive phenotype observed in 3D cultures. The differences in cell line invasiveness and related pathway regulation for basal subtypes could be specific to the selected culture environment. Future studies using a 3D environment without the presence of an external extracellular matrix such as ultra-low attachment plates or spinner vessels would reveal the effects of the matrix composition on pathway regulation and cell phenotype.We observed that cell lines of the basal A subtype showed a clear non-invasive spheroid morphology in 3D. In contrast, cell lines of the basal B subtype demonstrated a distinct invasive phenotype; an exception for the basal B phenotype was the SUM149PT, that shows ambiguous classification and has also sometimes been classified as basal A36. Next, proteasomal inhibition could disrupt essential cellular pathways resulting in cancer cell death 50. To investigate whether transcriptomic differences change drug sensitivity and define the optimal in vitro model for future drug screening; we systematically treated cell lines in both 2D and 3D cultures with specific inhibitors against CDKs, aurora kinases and the proteasome. Whereas previous studies mainly identified reduced sensitivity towards established drugs  in 3D cultures in mostly HER2 positive cell lines53, no change in sensitivity for CDKs, aurora kinases and proteasomal factors comparing 2D and 3D cultures was observed with our experimental setup. While the drugs that we tested were not evaluated in these studies, the discrepancies can also be explained by the differences in cell lines used and culture conditions. Additionally, we cannot exclude that limited differential sensitivity between 2 and 3D for the selected drug targets is related to limited changes of the target protein levels. For example, EGFR expression per se does not explain EGFR inhibitor sensitivity, and that EGFR inhibitors are effective in blocking EGFR auto-phosphorylation, without impacting on downstream signaling54. Hence, the TNBC proliferative and survival signaling wiring is complex and multiple pathways can contribute to overcome kinase inhibitor efficacy. Regardless, our data do indicate that across a broader panel of TNBC cell lines, typically 2D screening for the candidate drug targets will be as effective as 3D screening in identifying drug responses. To determine the optimal future screening model to assess efficacy of new targeted therapies, a thorough understanding of the overall clinical translation of the test system for specific cancer types, including TNBC, will be essential.CDKs, aurora kinases and proteasomal factors that were higher expressed in 2D, showed genuine higher expression levels in more aggressive breast tumors and cell lines, and were positively related to metastatic disease in breast cancer patients. Several aurora kinase inhibitors and Cell cycle-related CDK inhibitors have been developed of which some show promising results in clinical trials and are almost facing towards the clinicFor basal BC cell lines the transcriptome of 3D cultures demonstrated increased similarity towards human patient tumor transcriptomes, which was mainly caused by elevated levels of genes involved in extracellular matrix interactions. On the contrary, the transcriptome profile of luminal cell lines was already highly comparable to the patient tumor transcriptome in 2D conditions, but did not improve in 3D settings. Importantly, similarity of transcript levels was highly pathway and subtype dependent. Next to the pathway of interest, the ideal in vitro model might change depending on the BC subtype of interest. In general, pathways in triple-negative patient tumors are better correlated to basal cell lines than the same pathways in ER-positive tumors.Altogether, we can conclude that our systematic transcriptome analysis uncovered major differences between 2 and 3D models thereby increasing the global understanding of these in vitro test systems. Defining ‘the best in vitro model’ with efficient clinical translation is yet difficult and dependent on both the pathway and subtype of interest, although in general 3D cultures correlated better to human patient tumors. To support such a selection, we established an in silico web tool in to allow (1) differential expression of genes between 2 and 3D BC cell lines, (2) collect gene expression levels in different breast cancer subtypes (cell lines as well as patient tumor data) and (3) calculate correlation between different cell lines/culture conditions with human patient tumors (10.5281/zenodo.4560297).57. These models that are closer to the patient situation have higher costs and cannot yet be applied in similar throughput as 2D plastic models, but are recently widely exploited for their application in drug screening61. Future systematic comparisons of the transcriptome and proteome of all these test systems in comparison with large BC patient transcriptome and proteome datasets, and their response to candidate drug targeted therapeutic approaches will be essential to provide clarity about the most optimal pre-clinical in vitro test systems.We performed a systematic transcriptomic comparison between 2 and 3D BC cell lines. Although clear transcriptomic differences were identified between 2 and 3D culturing systems, additional proteomic analysis would add a layer of information that could help to validate the transcriptomic findings and more intensively discriminate between the different models. In this study, we did not include other in vitro cell models that are of relevance for BC anticancer therapy development, including BC tumor organoids, short-term patient-derived xenograft cell cultures, stem cell reprogramming and tumors-on-a-chip2 incubator. For 2D cultures, cells were grown on plastic tissue culture plates (Corning). For 3D cultures, cells were grown in a gel-mixture containing 6 mg/ml matrigel , 0.5 mg/ml collagen , 3.7 g/L NaHCO3 and 0.05 M HEPES.All cell lines were purchased from ATCC. Cells were grown in RPMI-1640 medium  supplemented with 10% FBS , 25 IU/ml penicillin and 25 µg/ml streptomycin (full RPMI) (ThermoFisher Scientific) at 37 °C in a humidified 5% COFor 2D cultures, cells were plated in a 6-well tissue culture plate  in full RPMI. RNA was collected 2 days after plating at 70% confluency using RNeasy plus mini kit (Qiagen) according to the manufacturer’s protocol.g), the upper aqueous phase was collected and RNA was precipitated by mixing with 600 μl isopropanol. Samples were incubated for 10 min at room temperature, followed by 15 min centrifugation at 4 °C  and washing with 1.2 ml 75% v/v ethanol. Samples were centrifuged for 5 min at 4 °C (7500 × g). Then the supernatant was removed and the pellet was air-dried and dissolved in 30 μl RNase free water.For 3D cultures, cells were plated in a 24-wells tissue culture plate . Wells were pre-coated with 200 μl matrigel  and cells were plated in a total volume of 400 μl. The following densities were used for the different cell lines: MDA-MB-231 (200 cells/μl), Hs578T (200 cells/μl), BT549 (200 cells/μl), SUM149PT (250 cells/μl), HCC1143 (250 cells/μl), HCC1806 (250 cells/μl), HCC1954 (250 cells/μl) and MDA-MB-468 (250 cells/μl). After 30 min incubation at 37 °C, 1200 μl full RPMI was added. RNA was collected 7 days after plating using the QIAzol method to reduce matrigel pollution. RPMI was removed, 1.2 ml QIAzol (Qiagen) was added per well and cells were lysed by repetitive pipetting. Cells were incubated for 5 min at room temperature and 240 μl chloroform was added, followed by 2–3 min incubation at room temperature. Samples were centrifuged for 15 min at 4 °C  developed by Dr. Richard Simon and the BRB-ArrayTools Development Team and R (v3.2.2). Genes with p-value < 0.01 were considered significant. Upon acceptance, RNA sequencing data will be available in the Sequence Read Archive.DNA libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit and sequenced according to the Illumina TruSeq v3 protocol on an Illumina HiSeq2500 sequencer. 100 base pair paired-end reads were generated and alignment was performed using the HiSat2 aligner (version 2.2.0.4) against the human GRCh38 reference genome. Gene expression was quantified using the HTseq-count software (version 0.6.1) based on the ENSEMBL gene annotation for GRCH38 (release 84). Count data was normalized using the DESeq2 package63 previously described by our group64.For 2D drug screening, cells were plated in a 96-well tissue culture plate  using the following cell densities: HCC1143 (8000 cells/well), HCC1806 , HCC1954 (8000 cells/well), MDA-MB-468 , Hs578T , MDA-MB-231 (5000 cells/well), SUM149PT (8000 cells/well) and BT549 . Drug treatment was performed 16 h after plating and 4 days after treatment, cells were fixed for sulforhodamine B (SRB) colorimetric proliferation assayFor 3D drug screening, cells were plated in a 384-well µclear plate  using a CyBi Selma 96/60 robotic liquid dispenser . The same gel composition and cell densities were used as in the RNA isolation procedure. Cells were plated in a total volume of 14.5 μl matrigel-collagen mix. After 30 min incubation at 37 °C, 44.5 μl full RPMI was added. Drug treatment was performed 3 days after plating and 4 days after treatment, cells were fixed and stained in 0.05 µM Phalloidin Rhodamin (Sigma Aldrich), 0.4 µg/ml Hoechst 33258 (Fisher Biotech), 0.1% Triton X-100 Triton (Sigma Aldrich) and 3.7% formaldehyde (Sigma Aldrich) in PBS O/N at 4 °C. Plates were washed with PBS and imaged using ImageXpress Micro XLS system .Both in 2D and 3D cultures, cells were treated with 6 concentrations of the following inhibitors: MG132, bortezomib, flavopiridol HCl, dinaciclib, palbociclib, milciclib, aurora kinase inhibitor I, TAK-901, gefitinib and erlotinib . DMSO was used as drug vehicle control.SRB absorbance values were used to analyze 2D cell line proliferation. To analyze the 3D cultures the acquired image stacks  were processed in OcellO Ominer 3D image analysis platform. The software processes the DAPI channel to measure the number of nuclei and the shapes of the nuclei in each image stack and integrates this information with the shape and number of tumor cell clusters detected in the TRITC channel. The software was used to process the total phalloidin area as measurement of proliferation.Proliferation was normalized to the absorbance (2D) or phalloidin area (3D) before treatment (set at 0%) and DMSO treatment (set at 100%). A nonlinear sigmoidal dose–response (with variable slope) curve was used to fit the data and retrieve the IC50 values using Graphpad Prism 7.00.For 2D immunofluorescence staining, cells were plated in a 96-well μclear plate (Greiner). Cells were fixed and permeabilized 72 h after plating by incubation with 1% formaldehyde and 0.1% Triton X-100 in PBS and blocked with 0.5% w/v BSA in PBS. Cells were incubated with 1:10,000 Hoechst 33258 combined with 1:2000 rhodamine-phalloidin  for 1 h at room temperature. Cells were imaged with a Nikon Eclipse Ti microscope and 20 × oil objective.65 using KEGG and Reactome databases. Ranked gene set enrichment analysis (GSEA) was performed on the full ranked gene lists66. T-sne plots were generated using the tsne R package. Hierarchical clusterings were generated using the pheatmap R package, based on complete linkage and Euclidean distance or correlations as described in the figure legend.Over-representation analysis was performed using Consensus PathDB67 after the new release in January 2017. Normalized reads were log2 transformed before further analyses were performed. For calculating spearman correlations between the TCGA and cell line RNA sequencing data, log2 normalized data were used for both datasets. Microarray data of primary tumors of 867 untreated patients (MA-867 dataset) used to calculate hazard ratios was previously published and publicly available . Raw .cel files were downloaded, processed with fRMA and batch effects were corrected using ComBat.Normalized total gene expression data from The Cancer Genome Atlas (TCGA) were obtained by using the TCGA Assembler R packageThis article does not contain any studies with human participants or animals performed by any of the authors.Supplementary Figure Legends.Supplementary Figures.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.