MYC and BCL2 and/or BCL6 resulting in MYC overexpression. Despite the poor prognosis of DHL, R-CHOP chemotherapy remains the treatment backbone and new targeted therapy is needed. We performed comprehensive cytogenetic studies/fluorescence in situ hybridization on DLBCL and Burkitt lymphoma cell lines (n = 11) to identify the DHL/THL DLBCL in vitro model. We identified MYC/IG in Raji and Ramos (single hit); MYC/IG-BCL2 (DHL) in DOHH2, OCI-LY1, SUDHL2, and OCI-LY10; MYC/IG-BCL2/BCL6 (THL) in VAL; and no MYC rearrangement in U2932 and HBL1 (WT-MYC). Targeting MYC in the DHL/THL DLBCLs through bromodomain extra-terminal inhibitors (BETi)  significantly (p < 0.05) reduced proliferation, similar to WT-MYC cells, accompanied by decreased MYC but not BCL2 protein. Moreover, BETi suppressed MYC transcription and decreased BRD4 binding to MYC promoter in DHL cells. CD47 and PD-L1 are immunoregulatory molecules often expressed on tumors and regulated by MYC. High levels of surface CD47 but not surface PD-L1 was observed in DHL/THL, which was reduced by JQ1 treatment. BETi in combination with Pan-HDAC inhibitor had a limited effect on survival of DHL/THL, while combination of BETi and BCL2 inhibitor (ABT-199) had a significant (p < 0.005) inhibitory effect on survival followed by BCL-XL inhibition. Overall, the data suggests that MYC-expressing DLBCLs are probably addicted to the MYC-oncogenic effect regardless of MYC rearrangements. In summary, we identified an in vitro model for DHL/THL DLBCLs and provide evidence for the therapeutic potential of BET inhibitor alone or in combination with BCL2 inhibitor.Double/triple-hit lymphomas (DHL/THL) account for 5–10% of diffuse large B cell lymphoma (DLBCL) with rearrangement of The online version of this article (10.1186/s13045-019-0761-2) contains supplementary material, which is available to authorized users. MYC rearrangements in DLBCL. Earlier studies reported that 5–15% of DLBCL harbored MYC, BLC2, and/or BCL6 translocations and were called “double-hit” lymphoma (DHL) or triple-hit lymphoma (THL). In the most recent WHO revision of lymphoma classification, DHL/THL category is now recognized as "high-grade B cell lymphoma (HGBL) with rearrangements of MYC and BCL-2 and/or BCL-6 [MYC rearrangements  co-occur with BCL-2 or BCL-6; however, in THL cases, MYC rearrangements  co-occur with both BCL-2 and BCL-6. The DHL with BCL-2 translocation has an aggressive clinical presentation and is hard to treat with conventional chemotherapy [BCL-6 cases (MYC/BCL6) is not well understood. With standard therapeutic approaches such as with rituximab, cyclophosphamide, doxorubicin, and vincristine (R-CHOP) [MYC and BCL2 genes are overexpressed at the protein level, without genetic rearrangements. MYC protein expression is detected in a much higher proportion of DLBCL (around 40%) and is associated with concomitant expression of BCL-2 [MYC and BCL-2 and/or BCL-6 [Diffuse large B cell lymphoma (DLBCL) is the most common aggressive B cell lymphoma in the USA. Based on gene expression profiling (GEP) studies, DLBCL can be classified into germinal center B cell (GCB) and activated B cell (ABC) subtypes . In addiotherapy , 4. The (R-CHOP) . There aof BCL-2 . This prof BCL-2 , 14. Theor BCL-6 , 14.Despite the poor prognosis in DHL, R-CHOP remains the backbone of treatment; it is an area of active preclinical and early-phase clinical research for exploring novel approaches for the treatment of difficult lymphomas. MYC and BCL2 translocations drive proliferation and prevent apoptosis in DHLs. We have previously shown that MYC overexpression correlated with inferior event-free survival in DLBCL . MYC actThe B cell lymphoma cell lines OCILY10 (LY10), SUDHL2 (DHL2) OCILY1(LY1), OCILy3, and OCILy19 were a kind gift from Dr. Louis Staudt . VAL and U2932 cell lines were kindly provided by Dr. Izzidore Lossos . All cell lines were grown in Iscove’s modified Dulbecco’s medium supplemented with 20% human serum and antibiotics/antimycotics. Raji, Ramos (BL), and DOHH2 cell lines were purchased from ATCC  and were cultured in RPMI supplemented with 10% FBS.Antibodies to c-MYC, BCL-6, BCL-2, BCL-XL, MCL-1, P21, BIM, and H3K27Ac were obtained from Cell Signaling Technology . Actin antibody was purchased from Santa Cruz . BET inhibitor I-BET762 (referred to as I-BET), JQ1, and OTX015 and BCL-2 inhibitor ABT-199 were purchased from Selleck Chemicals . HDAC inhibitor SAHA (vorinostat) was purchased from Sigma-Aldrich .MYC, BCL2, and BCL6 rearrangements were analyzed using break-apart FISH. The MYC (5′ red (R) /3′ green (G)), BCL2 (3′ G/5′ R), and BCL6 (3′ G/5′ R) probes were commercially available from Abbott Molecular . FISH was performed using standard FISH methodologies [dologies .4 cells were cultured for 72 h with bromodomain extra-terminal inhibitors (BETi). Before harvesting, cells were pulsed with 1 μCi (0.037 MBq) tritiated thymidine  for 18 h and 3H-TdR incorporation levels were determined using a Beckman scintillation counter (GMI). For XTT assay, 0.25 × 104 cells were cultured for 72 h with BETi and XTT was added for 3 h followed by analysis on a SpectraMax plate reader .For thymidine incorporation assay, 1.0 × 105 cells/ml were cultured for 72 h in the absence or presence of BET inhibitors, then stained using 1 μg/ml annexin V–FITC for 30 min at 4 °C. Cells were then washed in annexin V binding buffer and stained with 0.5 μg/ml propidium iodide and analyzed by flow cytometry . Data analysis was performed with Flow Jo software (TreeStar).5.0 × 10Cells were lysed with RIPA buffer for 30 min on ice and lysates cleared by centrifugation, and Western blotting was performed as described earlier .Total RNA was extracted using RNeasy mini kit . cDNA was synthesized using total RNA with SuperScript III First-Strand Synthesis SuperMix  according to the manufacturer’s instructions. PCR was performed according to the HotStar Taq Master Mix kit instructions. The program consisted of 95 °C for 15 min, 25 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s, followed by 72 °C for 10 min. The RT-PCR primers used were as follows:c-MYC: cMyc-F (5′GGGTAGTGGAAAACCAGCAGCCTC3′)cMyc-R (5′CATCTTCTTGTTCCTCCTCAGAGTCGC3′).BCL6: BCL6-F (5′TAACATCGTTAACAGGTCCATGACG3′)BCL6-R (5′GCCCCGTTCTCACAGCTAGAATC3′)GAPDH: GAPDH-F (5′GAAGGTCGGAGTCAACGG ATTTG3′)GAPDH-R (5′ATGGCATGGACTGTGGTCATGAG3′).6 DLBCL cells were transfected using a U-15 program on a Nucleofector equipment. Two days following transfection, cells were harvested and used for analyses as required.Plasmid DNA (5 μg) for each of the pcDNA3, pcDNA3-cMyc, or pcDNA3-BCL2 (addgene) was transfected using a human B cell Nucleofector kit (Amaxa Biosystems). Briefly, 6 × 10ChIP assay was performed using the ChIP Assay Kit  with antibodies to BRD4  or IgG following the manufacturer’s instructions. Immunoprecipitated DNA and input were analyzed by PCR using the following primers: c-MYC promoter: F: 5′-AACATGACCAGACTGCCTC-3′and R:5′-CTCAAAGCAAACCTCCTAC-3′; BCL6 promoter: F: 5′-CGTACATTCTCAGCTTATG-3′and R:5′-CTTACGCCTCTCTTTACTG-3′; and BCL2 promoter: F: 5′-CAAGGGGGAAACACCAGAATC-3′ and R:5′-CCCCCAGAGAAAGAAGAGGAG-3′.6 cells) were washed in FACS buffer (PBS containing 2% FBS and 0.05% sodium azide) and incubated with PD-L1-PE conjugated and CD47-FITC conjugated or isotype control antibodies (mouse IgGκ-FITC/PE)  for 30 min. Cells were washed with FACS buffer and re-suspended in 500 μL FACS buffer, and data were acquired on a FACSCalibur flow cytometer (BD Biosciences). Data was analyzed using FlowJo version10 software.Cells  seen in HBL-1 and U2932 cell lines; single MYC rearrangement with immunoglobulin commonly referred to as single hit  seen in Raji and Ramos Burkitt lymphoma cell lines; MYC rearrangement with BCL2 gene  seen in OCILY1, OCILY10, SUDHL2, and DOHH2 cell lines; or MYC rearrangements with both BCL2 and BCL6 genes  seen in only VAL cell line  using MYC, BCL2, and BCL6 break-apart (BA) probes. Break-apart probes target two areas of a MYC overexpression has been shown to be regulated by BRD proteins in multiple cancer types , 28. We Next, we increased the doses of JQ1, I-BET, and OTX to 2.5 and 5.0 μM and assessed the anti-proliferative effect in WT-MYC, MYC/IG, BCL2/MYC, MYC/BCL2/BCL6, and BCL2/BCL6 rearrangements. Raji cell line, which harbors a single MYC rearrangement, was the most sensitive to I-BET, JQ1, and OTX with nearly 95% inhibition of thymidine incorporation , SH (Raji), DHL , and THL (VAL) expressing human DLBCL lines were treated with various doses of I-BET, JQ1, or OTX. We consistently observed a potent, concentration-dependent decrease in MYC protein expression, across the panel of cell lines tested, suggesting that BETi suppress MYC regardless of MYC rearrangement status  showed robust MYC mRNA suppression by I-BET, JQ1, and OTX as compared to that observed in LY1 (DHL)  Fig. a, b. HowHL) Fig. a, b. TheMYC transcription by physically blocking binding of BRD proteins at regulatory elements that influence MYC expression. We analyzed the recruitment of BRD4 to the MYC promoter by ChIP assay and found that BRD4 was enriched at the MYC, BCL2, and BCL6 promoter regions  transcription potentially via decreasing BRD4 recruitment to the promoter region of MYC and BCL6.Mechanistically, BETi interfere with We extended the study of BET inhibition to examine the effect on survival of DHL/THL DLBCL cell lines. DLBCL cell lines expressing WT-MYC (U2932), SH (Raji), DHL , and THL (VAL) were treated with varied concentrations of I-BET, JQ1, or OTX015 and then analyzed for survival fraction of cells by excluding annexin V/propidium iodide-stained cells. Surprisingly, unlike cell proliferation data, BET inhibition had only a modest effect on cell survival for cell lines tested  and CD47 expression . We sougBETi mediated transcriptional repression of MYC or BCL6 appears to have little effect on survival of DHL or THL DLBCL cells despite robust anti-proliferative activity. Next, we sought to determine if BETi can sensitize cells to HDAC inhibition. WT-MYC (U2932), MYC/BCL2-(LY1), and MYC/BCL2/BCL6 (VAL) DLBCL cell lines were treated with or without suboptimal concentrations of SAHA, I-BET, or their combination for 72 h and analyzed for cell proliferation and survival. As shown in Fig. We next sought to determine the mechanisms of BETi and HDACi combination effect on cell proliferation of DHL and THL cells. We performed the western blot analysis in DLBCL cell lines harboring WT-MYC, MYC/BCL2, or MYC/BCL2/BCL6 rearrangements and tested the impact of combination on histone acetylation H3K27AC) and the cell cycle regulator P21. As expected, treatment with SAHA increased levels of H3K27 acetylation and P21, combining BETi and SAHA further increased the P21 level in WT-MYC, DHL, and THL cells  and MYC/BCL-2/BCL-6  cell lines. The combination of both drugs reduced BCL-XL and MCL-1 levels more than either single agent alone in WT and THL; however, no changes were seen in BIM levels at the doses used in all the cell lines tested Fig. d. OveralThe median overall survival of patients with DHL/THL DLBCLs treated with R-CHOP has been reported to be 5 to 24 months , and theThe major challenge for preclinical in vitro studies is the identification of DHL/THL DLBCL cell lines to be used as an in vitro model. We exploited and identified 4 DHLs  and 1 THL  along with WT-MYC (U2932 and Karpas 422) DLBCL lines by comprehensive cytogenetic analysis. These DHL/THL cell lines will serve as an in vitro model for preclinical studies. We further our studies by targeting BET protein through several small molecule bromodomain inhibitors including JQ1, OTX015, and I-BET-762 using DHL and THL DLBCL model cell lines. JQ1, OTX, and I-BET at lower concentration (0.05 or 1μM) had a minimum inhibitory effect on proliferation of DHL and THL cells, although at higher concentrations such as 2.5 μM or 5 μM all of the three inhibitors significantly abrogated the proliferation of DHL/THL DLBCL cells and the inhibitory effect was similar to that observed in wild-type MYC overexpressing DLBCL cells. Bhadury et al. have shown the anti-proliferative effect of JQ1 at a much lower concentration in DHL cells from transgenic MYC overexpressing mice . The difMYC+/BCL2+DHL model; however, this phenomenon was counteracted by the BET bromodomain inhibitor CPI203 [We found that pharmacological inhibition of BET proteins leads to no significant difference in cell survival of DHL, THL cells at the concentration it causes the growth inhibition. The selective anti-proliferative effect of BETi through MYC transcriptional program provides an opportunity to combine BET inhibitors with inhibitors of other signaling pathways. BET inhibitors have been shown to synergize AML cells to HDAC inhibitors . Our resr CPI203 .In summary, our findings provide a rationale for using BET bromodomain inhibitors alone or in combination with BCL-2-specific inhibitors as the first-line therapy option for double-hit and triple-hit patients. This study demonstrates that, apparently, there is no distinctive feature of DHL/THL lymphoma with respect to sensitivity to BET inhibitors, and MYC-expressing lymphoma cells are probably addicted to the MYC-oncogenic effect and rely on MYC for their growth regardless of MYC rearrangements. Moreover, our study emphasizes on incorporation of FISH analysis along with MYC expression into the standard diagnostic procedure for prompt and accurate identification of DHL and THL patients to start the BET inhibitor therapy alone or in combination with other therapeutic drugs to improve the outcome.Additional file 1:Figure S1. Effect of low doses of I-BET on the proliferation of DHL/THL DLBCL cells. (A-B) WT-MYC, SH, DHL, THL, and BCL2/BCL6 translocation harboring DLBCL cell lines were treated with low doses of I-BET (0.5 μM and 1.0 μM) and proliferation (A) and survival (B) analyses was performed. (TIFF 1142 kb)


H). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay–a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703–21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRI H) chain, as the rat homologue receptor does not bind human IgE with high affinity ; in et al. [H) surface expression is due to different levels of expression of the mRNA encoding the human gamma chains (FcεRIγH) between the different humanised cell lines, resulting in different levels of competition between the endogenous rat and the introduced human gamma chains at the protein level for assembly into the tetrameric receptor complex.The humanized RBL703/21 and NFAT-DsRed, which was derived from the former, are single α-chain transfectants. This fact might explain why the surface levels of the human FcεRI α-chain are lower in these two humanized RBL cell lines than the SX-38 and RS-ATL8. Taudou et al.  suggesteb significantly increased surface expression of the α-chain, with or even without the γ-chain. Once the FcεRI receptor has reached the surface, the binding of exogenous IgE stabilizes it [Finally, in terms of posttranslational regulation of FcεRI expression, work by Platzer and co-workers  suggestelizes it . All traH expressing cell lines.The observation that, in the absence of any sequence differences in the coding region of the α-chain between the four cell lines  or diffeH-chain, the NFAT-DsRed responds well to IgE-dependent stimulation, as it was derived by two rounds of FACS sorting followed by cloning, resulting in a highly reactive clone. The twice-sorted clone has a 8-fold higher sensitivity than the single sorted and a signal to noise ratio of ~30fold  of allergens in array (or multiwell format) at a lower cost. Therefore, our future efforts will be directed at obtaining a fluorescent humanized reporter assays with FcεRIαH expression levels comparable to the RS-ATL8.Despite the lower FcεRIαS1 FigExample of PCR amplification of human FcεRIα (A) and GGT1 gene (B) from parental RBL-2H3 cells, from which all humanized cell lines described in this paper were derived. Genomic DNA was extracted from RBL-2H3 cells and subject to PCR amplification as described in Materials and Methods using the gene of interest (HsFcεRI) primers (A) and the reference gene (rat GG1) primers (B). As expected, there is no amplification with the human FcεRIα-specific primers, demonstrating lack of amplification of the endogenous homologous FcεRI rat gene.(PDF)Click here for additional data file.S2 FigA) Map (SnapGene) of the coverage obtained from sequencing the cDNA with 6 different primers. The binding positions and names of the primers are indicated at the top of the map in purple. The bottom of the map also indicates the positions of the signal sequence peptide, the transmembrane domain and the 5 ER retention signals (RS) D192, K212, K216, K226 and K230. Full multiple coverage was obtained for all four humanized RBL cell lines, demonstrating complete identity of the cDNA sequences. B) Details of the chromatograms in the region containing the five known retention signals.(PDF)Click here for additional data file.S3 Figfl microscope at 100x magnification using the RFP light cube.After a further incubation of 16–18 hours, responding cells producing DsRed were sorted by flow cytometry as single cells into 96-well plates, and clones allowed to grow and expand for several weeks. The highest responding cells were pooled and the process, consisting of activation, sorting and cloning was repeated once more. Individual 2x sorted clones were expanded and tested for their response to anti-IgE. A) shows the response of the 1x sorted cells, a 2x sorted high responding clone (A6) and an intermediate responding clone (H8) to activation via the IgE receptor (2 μg/mL anti-IgE) or 1μg/mL A23187. After removal of the medium, cells were lysed in 1% v/v Triton X-100 in DPBS and the lysate transferred to low-autofluorescence black plates. Fluorescence was read in an Infinite M200 plate reader , using 530nm excitation and 590nm emission filters . B) shows the same cells with and without IgE-dependent activation under the EVOS (PDF)Click here for additional data file.


In vitro drug-resistant cell model would help in the understanding of molecular mechanisms underlying this drug-resistance phenomenon. The aim of this study was to characterize new models of CDDP-resistant GC cell lines (AGS R-CDDP and MKN-28 R-CDDP) obtained through a stepwise increasing drug doses method, in order to understand the molecular mechanisms underlying chemoresistance as well as identify new therapeutic targets for the treatment of GC. Cell viability assays, cell death assays and the expression of resistance molecular markers confirmed that AGS R-CDDP and MKN-28 R-CDDP are reliable CDDP-resistant models. RNA-seq and bioinformatics analyses identified a total of 189 DEGs, including 178 up-regulated genes and 11 down-regulated genes, associated mainly to molecular functions involved in CDDP-resistance. DEGs were enriched in 23 metabolic pathways, among which the most enriched was the inflammation mediated by chemokine and cytokine signaling pathway. Finally, the higher mRNA expression of SERPINA1, BTC and CCL5, three up-regulated DEGs associated to CDDP resistance found by RNA-seq analysis was confirmed. In summary, this study showed that AGS R-CDDP and MKN-28 R-CDDP are reliable models of CDDP resistance because resemble many of resistant phenotype in GC, being also useful to assess potential therapeutic targets for the treatment of gastric cancers resistant to CDDP. In addition, we identified several DEGs associated with molecular functions such as binding, catalytic activity, transcription regulator activity and transporter activity, as well as signaling pathways associated with inflammation process, which could be involved in the development of CDDP resistance in GC. Further studies are necessary to clarify the role of inflammatory processes in GC resistant to CDDP and these models could be useful for these purposes.Gastric cancer (GC) is a significant cancer-related cause of death worldwide. The most used chemotherapeutic regimen in GC is based on platinum drugs such as cisplatin (CDDP). However, CDDP resistance reduces advanced GC survival.  Gastric cancer (GC) is the sixth most common malignancy and the third leading cause of cancer-related deaths in both sexes in the world, making it a major global public health problem . As mostin vitro models of acquired or induced drug resistance is a useful approach to better understand the mechanisms that trigger clinical resistance to chemotherapeutics. In addition, in vitro models can clarify the cellular and molecular mechanisms of novel anticancer agents, enabling comparisons with parental cells and intrinsically resistant cells [The development of more reliable nt cells . The aimThis study was approved by Ethical Committee of Universidad de La Frontera .Cisplatin (CDDP) was purchased from Selleck Chemicals . CDDP was reconstituted at a concentration of 3.3 mM diluted in 0.9% (p/v) NaCl and aliquots of stock solution were stored at −80°C.2 conditions. Cells were subcultured at 80% confluence and harvested after treatment with 0.25% trypsin and 0.02% EDTA .AGS and MKN-28 cell lines were generously provided by Dr. Richard Peek . AGS was established from a gastric adenocarcinoma obtained from a 54-year-old female  and MKN-50 concentration. Cells were seeded according to doubling time, and the starting dose of the drug was incorporated into the cells when they presented 20% confluence. The increase in drug doses was made every two subcultures, by doubling each previous concentration. The cycle was repeated 30 times. Once cells acquired cisplatin resistance they were grown in drug-free medium for one month, frozen in liquid nitrogen and then awakened in medium containing CDDP to confirm the level of drug resistance. The time for the development of this drug-resistant model was 12 months.Induced drug-resistant cell lines, CDDP-resistant AGS cells (AGS R-CDDP) and CDDP-resistant MKN-28 cells (MKN-28 R-CDDP) were developed following Coley’s protocol . Briefly3 for parental cells and 5.5x103 for resistant cells according to their doubling time) in 100 μL of culture medium and incubated for 24 H to allow cell attachment and to reach a 50% confluence. Next, cells were exposed for 72 H at different concentrations of CDDP, ranging from 0.01 μM to 1000 μM. Cells without CDDP were used as controls. After 72 H of incubation the medium was removed, and cells were washed with 100 μL of DPBS/Modified . Then, 0.5 mg/mL of MTT was added to each well, followed by 2 H incubation. As only functional mitochondrial dehydrogenase enzymes from viable cells can reduce MTT to form formazan, 100 μL of propanol was used to fully dissolve this purple precipitate. Absorbance was measured at 570 nm using the Infinite® NanoQuant spectrophotometer . The EC50 values (drug concentration that inhibited cell growth at 50%) were estimated through the dose-response curve after 72 H of incubation under different drug concentrations. In this case, the percentage of viable cells was plotted according to the corresponding drug concentrations, obtaining the values of half maximal effective concentration (EC50) by non-linear regression. The resistance index (RI) values were calculated by dividing the EC50 values of resistant cell lines by the EC50 values of parental cell lines, defining arbitrarily the chemoresistance as RI ≥2.Drug sensitivity analyses were performed using a standard viability assay (MTT assay). Briefly, cells were seeded in 96-well plates (4x10™ 488 annexin V and propidium iodide (PI) (Invitrogen) according to the manufacturer’s instructions. Briefly, parental and resistant cells were seeded in 6-well plates  in 2 mL of culture medium and incubated for 24 H to allow cell attachment. Then, cells were treated for 72 H at EC50 concentrations of resistant cells . Following incubation, cells were harvested by trypsin and the pellets were centrifuged, washed with 1X PBS and resuspended in 100 μL of 1X annexin buffer. For staining, cells were incubated with 5 μL of Alexa Fluor® 488 annexin V and 1 μL of 100 μg/mL Propidium iodine (PI) at 37°C for 15 minutes. Finally, cells were resuspended with 400 μL of 1X annexin buffer and collected for analysis by flow cytometry . 5% of DMSO was used as an apoptosis-inducing agent. Early apoptotic cells , late apoptotic cells (annexin V-positive and PI-positive) and annexin V-negative and PI-positive cell populations were all considered dead cells. The fluorescence was read at maximum excitation/emission of 499⁄521 for Alexa Fluor® 488 annexin V and 535⁄617 for PI.A cell death assay was performed using the Dead Cell Apoptosis Kit with Alexa FluorABCC2, ATP7A and CTR1 genes), as well as the expression profile of genes obtained by subsequent RNA-seq analysis  were performed by qRT-PCR. Total RNA was extracted from ~2.0 x106 cells using TRIzol Reagent  according to the manufacturer’s instructions. RNA concentration and integrity were evaluated at 260 nm using the Infinite®NanoQuant spectrophotometer  and by gel electrophoresis. Then, RNA was treated with DNase I  and first-strand cDNA was prepared from 2 μg of RNA in a total reaction volume of 20 μL using M-MLV reverse transcriptase 200 U/μL  at 42°C for 60 min. Subsequently, cDNA was amplified by qPCR using Brilliant II Ultra-Fast SYBR® Green qPCR Master Mix according to the manufacturer’s protocol in the Stratagene Mx-3000p real-time PCR system . Relative expression was determined using the 2-ΔΔCT method, using ACTB as the reference gene. Sequences of oligonucleotides used in this study are detailed in The expression profile of molecular markers involved in CDDP resistance  according to the manufacturer’s instructions. RNA integrity was checked by Agilent Bioanalyzer 2100 with a RNA Integrity Number (RIN) value ≥ 7.0. cDNA libraries were prepared according to KAPA Stranded RNA-Seq Kit  with the RiboErase system for ribosomal RNA depletion. Finally, the library´s quality and quantity were analyzed using the Agilent Bioanalyzer 2100 and Life Technologies Qubit3.0 Fluorometer, respectively. 150 bp paired-end sequencing was performed on an Illumina HiSeq 4000  instrument. The quality control (QC) of raw reads was checked with the FastQC tool [https://osf.io/ck39n/. Two biological replicates per cell line type were evaluated by RNA-seq analysis. Overexpressed genes were validated using qPCR (as described before) and western blot analyses comparing parental and CDDP-resistant cell lines. The sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession number PRJNA591481.Total RNA was isolated from ~2.0 x10tQC tool . RNA-seqtQC tool –20. FirstQC tool . The humtQC tool . After rtQC tool . A P-valGene ontology (GO) annotation and signaling pathway analysis of DEGs between CDDP-resistant and parental cell lines, were analyzed using the PANTHER Classification System (version 14.0)  and Meta™ Imager  according to the manufacturer’s instructions, and then quantified by densitometry using a ChemiGenius Gel Bio Imaging System .Parental and resistant cellular pellet was obtained at 70% of confluence after three subcultures. Subsequently, protein lysates were harvested using RIPA buffer  containing protease  and phosphatase  inhibitors. The protein concentrations were determined using a bicinchoninic acid assay . An amount of 40 μg of the proteins were separated by SDS-PAGE and transferred  to PVDF membranes . Human SERPINA1, BTC and CCL5 protein expression levels were quantified using goat monoclonal antibody specific for human SERPINA1 and BTC  and goat polyclonal antibody specific for CCL5 protein . The expression levels of these three proteins were standardized to human β-actin using a rabbit monoclonal anti- β-actin antibody . Primary antibodies were detected using donkey anti-goat or goat anti-mouse or mouse anti-rabbit- radish peroxidase (HRP)-conjugated secondary antibodies . Immunoreactive bands were visualized using myECL50 values were calculated from dose-response curves using non-linear regression. P<0.05 values were considered statistically significant with 95% confidence interval.All the experiments were performed in biological and technical triplicates for each condition. Data were analyzed using the GraphPad Prism 5.0 software . Cell viability, cell death assay, qPCR and protein levels data were analyzed using the Mann-Whitney test. The EC50 values. The degree to which CDDP induced resistance was estimated as the ratio of the EC50 of each resistant line with its respective parental cell lines. EC50 values and RI are summarized in Viability assays made it possible to obtain dose-response curves  and non-Analysis of cell death by flow cytometry using annexin V and PI staining showed significantly lower cell death rates in CDDP-resistant cells than parental cell lines. When overall death percentages  were compared, AGS R-CDDP cells showed a statistically significant reduced cell death percentage (14.5%) compared with parental AGS cells (47.5%) P<0.001; . RepreseSimilarly, MKN-28 R-CDDP cells showed a statistically lower cell death percentage  than parental MKN-28 cells  P<0.01; . RepreseABCC2, ATP7A and CTR1 were evaluated on the AGS and MKN-28 cell lines subjected to both conditions: parental and CDDP-resistant cells  and their parental cells (AGS and MKN-28 cells without drug exposure). A total of 189 genes were found significantly differentially expressed (P<0.05 and fold change ≥2); 178 genes were up-regulated, and 11 genes were found down-regulated in CDDP-resistant cells compared to parental cells .binding (32%), catalytic activity (32%), molecular transduces activity (11%), transcription regulator activity (10%), transporter activity (6%), molecular function regulator (6%) and structural molecular activity (3%)  . Among tinflammation mediated by chemokine and cytokine signaling pathway (16%), integrin signaling pathway (11%) and interleukin signaling pathway (11%). Within inflammation mediated by chemokine and cytokine signaling pathway, 6 up-regulated genes were enriched; ITGAL, ITGAM, FPR1, CCL5, AP000295 and ALOX5AP. Within the integrin signaling pathway, 4 up-regulated genes were enriched: ITGAL, ITGAM, COL4A4 and LAMA2. Finally, within the interleukin signaling pathway, 4 up-regulated genes were enriched: IL3RA, IL15RA, AP000295, CSF2RB.  and BTC  of AGS R-CDDP and MKN-28 R-CDDP cells  confirmed that these cells presented a significantly greater tolerance to higher CDDP concentrations compared to parental counterparts [in vitro and in vivo studies, a minimum threshold of 2-fold of resistance has been widely accepted in clinical studies because responses have been observed using the double of the standard clinical dose of CDDP in drug-intensive therapy protocols [As expected, the ECterparts . Althougrotocols . Anotherrotocols , which dCell death assays showed that the CDDP-resistant cell lines AGS R-CDDP and MKN-28 R-CDDP had significantly lower cell death rates than their parental cell lines. These results agree with numerous studies showing that tumor cells resistant to chemotherapy escape cell death to protect against antitumor treatment –38. In tABCC2, CTR1 and ATP7A. ABCC2, member 2 of the ATP-binding cassette (ABC) transporter subfamily C, has been previously implicated in CDDP resistance because is one of the main transporters involved in the efflux of this drug [in vitro and in vivo [ATP7A, a copper transporting P-type ATPase, has been also implied in CDDP resistance in cancer [CTR1 gene was downregulated in AGS R-CDDP and MKN-28 R-CDDP cells, suggesting that CTR1 might be involved in CDDP resistance in both cell lines by decreasing the influx of this drug. These results were concordant with those obtained in CDDP-resistant A2780 human ovarian carcinoma cell line (A2780cis), which showed decreased CDDP accumulation and lower CTR1 expression compared to parental A2780 cell line [CTR1 mRNA found in ovarian tumors were associated with poor clinical outcome [ABCC2, which was concordant with the ABCC2 overexpression related to the drug resistance contribution in mammalian cells [ABCC2 was found down-regulated. On the other hand, ATP7A showed a significant decrease in MKN-28 R-CDDP cells but not in AGS R-CDDP where non-statistical differences were observed between CDDP-resistant and parental cells. Although higher ATP7A levels mediate platinum drugs resistance in different cell lines [ATP7A expression have been previously found in ovarian tumors prior and after treatment, thus additional studies are needed to validate this gene as a molecular marker of resistance [Regarding relative expression of molecular markers involved in CDDP resistance, three molecular markers were used to characterize each model cell; his drug ,40. The his drug ,42, and  in vivo . On the n cancer ,44 by inn cancer . In thisell line . In addi outcome . Only AGan cells ,49, meanll lines ,51, the sistance ,52.binding, catalytic activity, molecular transduces activity, transcription regulator activity, transporter activity, molecular function regulator and structural molecular activity. As drug resistance is a multifactorial process, many of these molecular functions such as binding, catalytic activity, transcription regulator activity and transporter activity are closely related to the platinum-drug resistant phenotype [Once the CDDP-resistant cell lines were established, a transcriptome analysis was performed to identify the DEGs between CDDP-resistant cells and parental cells. The RNA-seq analysis showed 189 DEGs of which 139 were involved in molecular functions including henotype ,54.Binding category has been previously described as a principal molecular function in several transcriptomic analyses. Ahn et al. performed RNA-seq analysis in melanoma models of resistance: BRAF inhibitor-sensitive A375P BRAF V600E cells, BRAF inhibitor-resistant counterparts (A375P/Mdr), and SK-MEL-2 BRAF-WT cells with intrinsic resistance to BRAF inhibitors. These analyses showed that the most DEGs were enriched in binding category  [et al. showed that a high proportion of DEGs found between tamoxifen-sensitive and tamoxifen-resistant MCF-7 breast cancer cell line belonged to the binding subcategories  [et al. also found a high proportion of DEGs within the binding category  when CDDP-resistant lung adenocarcinoma cell line A549 was compared to its parental A549 cell line [0 genes) . Similarll DEGs) . MoreoveCatalytic activity was another of the most enriched functions in GO analysis, which also presented the hydrolase activity and oxidoreductase activity as its main subcategories. When CDDP enters into the cytoplasm is activated by a series of hydrolysis reactions carried out preferentially by thiol-containing molecules, such as glutathione (GSH) and metallothionines [hionines ,57. An ihionines . CDDP fohionines ,59.transcription regulator activity. Transcription factors contribute to drug-induced responses and can induce either transient or acquired drug resistance [NRA42 , an orphan nuclear receptor that confers chemoresistance in GC and colorectal cancer [Another of the most enriched molecular functions in the GO analysis was sistance . Many trl cancer ,62.transporter activity category, particularly with transmembrane transporter activity. Membrane transporter proteins such as ATP7A, ATP7B [CTR1 role [ATP9A and ATP10A were differentially expressed. Because ATP-dependent efflux protein, is one of the main mechanisms responsible for multi-drug resistance (MDR) [On the other hand, as alteration of drug flux (increased efflux and/or decreased influx) is one of the principal mechanisms of drug resistance. Decreased drug accumulation is a consistent feature of many CDDP-resistant cell lines, mainly attributed to membrane transporter proteins . In thisA, ATP7B  could coTR1 role . In our ce (MDR) , these tinflammation mediated by chemokine and cytokine signaling pathway, integrin signaling pathway and interleukin signaling pathway. Cytokines are low molecular weight proteins involved in the induction and regulation of interactions of the immune, inflammatory and hematopoietic systems [ITGAL and ITGAM) are involved in two signaling pathway, such as inflammation mediated by chemokine and cytokine signaling pathway and integrin signaling pathway. The interleukin receptor AP000295 has been found to participate in both the inflammation mediated by chemokine and cytokine signaling pathway and interleukin signaling pathway. Therefore, this background not only suggest that the resistant cell lines have been undergoing a pro-inflammatory process but also that CDDP resistance can be enhanced by these pro-inflammatory mechanisms.According to our results, three signaling pathways were mainly involved in our CDDP-resistant models:  systems ,65. With systems . In addiIL6) and interleukin-8 (IL8) in resistant cell lines) [IL-6 secreted by CAFs is a critical contributor to chemoresistance in GC cells through the activation of the Jak1-STAT3 signaling pathway [NFκB (Nuclear Factor Kappa B) and TNF (Tumor Necrosis Factor) have also been associated to drug resistance [Several studies have focused their attention on the regulation of multi-drug resistance during an inflammatory response, in particular by pro-inflammatory cytokines . Cytokinl lines) . Evidencl lines) . In this pathway . On the sistance . Despitesistance .SERPINA1 (α-1-antitrypsin), BTC (Probetacellulin) and CCL5 (C-C motif chemokine ligand 5), three up-regulated genes associated to CDDP resistance according to RNA-seq analysis, was confirmed in CDDP-resistant cells compared to their parental counterparts. SERPINA1 and BTC belong to the top five differential expressed genes associated to CDDP resistance, meanwhile that CCL5 was involved in the most enriched metabolic pathway; inflammation mediated by chemokine and cytokine signaling pathway.Finally, the higher mRNA expression of SERPINA1, BTC and CCL5 are involved in molecular functions related to drug resistance. SERPINA1 was involved in molecular functions such as binding, catalytic activity and molecular function regulator; BTC was implied in molecular functions including binding, molecular transducer activity and molecular function regulator, meanwhile CCL5 was also involved in all the molecular functions described above.GO analysis confirmed that SERPINA1 is a serine protease inhibitor involved in endopeptidase inhibitor activity, protease binding and serine-type endopeptidase activity. Higher levels of SERPINA1 has been associated with chemoresistance of human epithelial ovarian cancer [SERPINA1 in resistance to CDDP in GC has not been clarified yet.According to PANTHER Classification System, the protein encoded by n cancer  and brean cancer  and has n cancer . HoweverBTC is a member of epidermal growth factor (EGF) family involved in cell proliferation, epidermal factor receptor binding and positive regulation of cell proliferation [BTC has been previously associated with worse survival in GC [BTC gene with CDDP resistance in GC cancers.On the other hand, feration . High exal in GC ; howeverCCL5 also known as Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES), is a chemokine secreted in a paracrine or autocrine fashion which has been associated with enhance the cancer progression in multiple myeloma (MM), classical Hodgkin lymphoma (cHL), prostate, breast, gastric, colon, and ovarian cancer, and melanoma [CCL5 has been shown to be an inductor of resistance to taxane [CCL5 role in the development of chemoresistance for any of the drugs commonly used for GC has not been evaluated.Besides, melanoma . During o taxane , CDDP [7o taxane , tamoxifo taxane  and Src o taxane  in modelSERPINA1 and BTC showed a differential expression of mRNA profile between parental and CDDP-resistant cells, as shown in the literature. Meanwhile CCL5, only showed a differential expression of mRNA profile between parental and CDDP-resistant AGS cells, which can be explained for the differences in molecular background between AGS[In this work, tween AGS and MKN-tween AGS cells.SERPINA1), meanwhile CCL5 protein could not be detected reliably by western blot analysis. According The Human Protein Atlas [SERPINA1 and CCL5 can undergo post-transcriptional regulation related with miRNA, like mir-940 [et al [Regarding protein levels, no differences were observed for α-1-antitrypsin  and new molecular markers of CDDP resistance .Characterization studies have effectively demonstrated that AGS R-CDDP and MKN-28 R-CDDP are reliable models of CDDP resistance because resemble the resistant phenotype of GC and show a significant higher tolerance to increasing concentrations of CDDP. These features were confirmed by significant differences in the RNA expression of known resistance markers  Metascape enrichment clustering analysis shows the P-value statistical significance among the different GO terms. B) Genes involved in three principal signaling pathways.(TIFF)Click here for additional data file.


All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment. While breast cancer is commonly represented as one disease, it is highly heterogeneous. Many studies have observed molecular heterogeneity, which is associated with distinct drug responses and clinical outcomes ,3,4,5,6.All-trans retinoic acid (atRA) is clinically used in the treatment of acute promyelocytic leukemia (APL) and high-risk neuroblastoma ,13,14, aRetinoid-based therapies have low systemic toxicity when compared to conventional chemotherapeutics, and the side-effects of atRA in treatment of APL are predominantly attributed to the differentiation of promyelocytes . This poWe validate our predictive DNA methylation profile in predicting the response of four TNBC patient-derived xenografts (PDXs) to systemic atRA treatment. By adding the PDXs to the model, we further refine the CpG profiles used to determine sensitivity and demonstrate a potential impact on as many as 17% of TNBC patients. This provides a paradigm for the utilization of differential DNA methylation as a predictive indicator of drug response. Our work demonstrates that atRA may be a clinically relevant choice for patients with TNBC and supports further consideration of atRA as a therapeutic option for TNBC.scid mice and observed a range of effects on tumor volumes and weights  in the genome, as opposed to retinoic acid response elements (RAREs) ,38. In cExpression and methylation of RARβ2  is anothConsistent with our previous data ,41, we cFor a more comprehensive evaluation, we similarly profiled the expression of other retinoid processing and signaling genes . When atRA-sensitive cell lines were compared to all other TNBC cell lines profiles, we identified 170 transcripts which were differentially expressed at basal levels ; 26 tranp-value < 0.01) demonstrate that many of the classic RA target genes such as DHRS3, CYP26A1, and STRA6, although not induced in every cell line, are induced in many of the cell lines.Furthermore, considering that few canonical atRA-inducible genes were identified in this comparative analysis (sensitive versus other cell lines), we confirm that the microarray analysis identified numerous classical RA target genes in analyses performed on individual cell lines . Cut-offWe have previously demonstrated that DNA methylation contributes to both baseline expression and the inducibility of atRA-responsive genes ,33,41. TUsing the Affymetrix HuGene 2.0ST data for PDX A and B, we first characterized the intrinsic subtypes using the single-sample predictor and the claudin-low algorithm. PDX A and B were classified as basal-like, and PDX C and D have been previously identified as basal-like . BaselinWe next investigated whether DNA methylation of tumor cells (sorted as demonstrated in scid mice with slow release atRA pellets significantly decreased tumor volume and tumor weight of four PDXs (p < 0.01) between all sensitive cell lines and PDXs and all other cell lines. The top six probes were identified as associated with genes TBCD, CCDC112, KHDRBS2, CD97, DPF3, and LRP5  in glioma predicts better response to alkylating agents . HoweverConsidering that cell-line xenografts may have limited clinical relevance and that the in vitro biomarker discovery may not be entirely transferable to in vivo usage, we utilized four PDXs in this study and demonstrate potential benefit of atRA treatment. However, there is limited heterogeneity in the PDXs used . Although basal-like tumors represent the majority of TNBCs , the incWith the exception of SUM149 and SUM159 cells that were obtained from BioIVT , all other cell lines were obtained from the American Type Culture Collection (ATCC) and maintained as recommended by supplier. Where indicated, all-trans retinoic acid  was used at 100 nM for 18 h.scid mice . Primary tumor volume was quantified  for the duration of the experiment. Tumor-bearing mice were euthanized and tumor weight quantified.One day prior to tumor-cell implantation, experimental mice were implanted with a slow-release atRA pellet . Cells were admixed 1:1 with high concentration Matrigel and injected orthotopically into the mammary fat pad of female NOD-3 pieces were sutured to the thoracic mammary fat pad of female NOD-scid mice. After palpable tumors developed, mice were euthanized, tumors harvested, and 2–3 mm3 pieces were successively implanted.PDX A and B were derived from patients at the QEII Health Sciences Center . Surgical biopsies from primary tumors were harvested and stored in DMEM with 10% FBS for <1 h until implantation. 2–3 mmscid mice for expansion, and subsequently preserved in liquid nitrogen.PDX C (BCM-3887) and PDX D (BCM-2665) were obtained from the laboratory of Dr. Michael Lewis  as frozen samples . Upon reTotal RNA was extracted using Trizol reagent and the PureLink RNA kit  with DNase treatment. Equal amounts of RNA were reverse-transcribed using iScript , and relative real-time PCR (qPCR) was performed using gene-specific primers . Standarg. Cells were resuspended in red blood cell lysis buffer . After 5 min, cells were centrifuged, resuspended in PBS, and passed through a 70 µm strainer. Cells were centrifuged, resuspended in Aldefluor buffer , and passed through a 70 µm strainer. Approximately 1 × 107 cells were incubated with anti-H-2Kd  at 37 °C with shaking. After 1 h, cells were centrifuged and resuspended in Aldefluor buffer with 7-AAD . Stained cells were gated on SSC and FSC to eliminate doublets. 7-AAD- H-2Kd- cells were sorted into ice-cold PBS with 5% BSA (Sigma-Aldrich). H-2Kd purity was assessed .PDX-bearing mice were euthanized, and tumors harvested. Tumors were minced and incubated in collagenase  in HBSS, Thermo Fisher Scientific, Mississauga, ON, Canada) at 37 °C on an end-over-end shaker. After 2 h, cell suspension was passed through a 70 µm strainer (Fisher Scientific) and centrifuged for 5 min at 500× TNBC cells were treated with atRA; total RNA was extracted as described. Sample preparation, amplification, hybridization to the Affymetrix HuGene 2.0 ST array, and data collection were performed by The Centre for Applied Genomics at the Hospital for Sick Children  and data is accessible from GSE117579. HuGene 2.0 ST data for MDA-MB-231 and MDA-MB-468 cells with and without atRA treatment was obtained from GSE103426 . ExpressHM450 data for all cell lines was obtained from GSE78875 , and anaDNA was extracted from human cells of PDXs using the PureLink DNA kit (Thermo Fisher Scientific), according to manufacturer’s instructions. Sample preparation, bisulfite conversion, hybridization to the Illumina EPIC array (PDX C and D), and data collection were performed by The Centre for Applied Genomics (TCAG) at the Hospital for Sick Children . Data can be accessed through the GEO repository (GSE117926). HM450 data (PDX A and B) was obtained from GSE78875 . HM450 aRNA was extracted from all cells of PDXs using Trizol reagent and the PureLink RNA kit (Invitrogen) with DNase treatment. Sample preparation, labelling, and hybridization to the Affymetrix HuGene 2.0ST array was performed by TCAG and data can be accessed through the GEO repository .PDXs were subtyped with normalized RNA expression using SSP2003 via the genefu package in the R environment ,67. The Data from TCGA  was extrt-test. The molecular subtype and the presence of alterations in driver genes were compared between sensitive cell lines and all remaining cell lines by a Fisher’s exact test. For all analyses, * p < 0.05, ** p <0.01, *** p < 0.001.All statistical analyses beyond those described in the R environment were conducted with GraphPad Prism 6.0. Tumor weights were compared by student’s All experiments were conducted in accordance with the Canadian Council on Animal Care standards and protocols approved by Dalhousie University Committee on Laboratory Animals (#15-013 and #17-011). PDX A and B patient samples were collected, maintained, and analyzed in accordance with protocol #1007106, approved by the IWK Health Centre Research Ethics Board.We detail a methylation-based predictive profile to identify atRA sensitivity in TNBC. Importantly, if proven successful for atRA, it may be possible to use a similar differential DNA methylation profiling strategy to predict sensitivity to other drugs, including more potent and selective retinoids and rexinoids. Hence, these findings provide a paradigm for utilizing DNA methylation as a stratification approach for treatment selection.


The hybrids soaked in simulated body fluid (SBF) showed the formation of hydroxyapatite layers on their surface, suggesting that the hybrids are bioactive. Their radical scavenging capacity towards DPPH· and ABTS·+ , evaluated at three different doses , showed probe- and dose-dependent behavior. In addition, the antioxidant properties of CGA were not affected by the presence of high amounts of the polymer. The in vitro biocompatibility in three cell lines  was assessed by using the 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apart from SH-SY5Y, the cell viability—expressed as mitochondrial redox activity percentage of cells directly exposed to powders—and morphology was not affected, suggesting that the hybrids have the ability to interfere and act selectively against tumor cells. The antibacterial properties of the different materials against Escherichia coli and Enterococcus faecalis were affected by different amounts of the natural antioxidant component.The formation of pro-oxidant species after implantation of biomaterials could be responsible for the failure of the implant itself, because of oxidative stress-induced damage. In this work, the SiO Under physiological conditions within the human body, cells are able to counteract the production of free radicals thanks to various enzymes that manage the redox homeostasis. However, when the biological repairing mechanisms are not sufficient and/or pro-oxidant species are overproduced, the homeostatic processes fail and the pro-oxidative/antioxidative cellular balance is compromised. In this condition, called ‘oxidative stress’, reactive oxygen species (ROS), nitrogen species (RNS) and consequently lipid peroxidation (LPO) products promote cellular injury and tissue damage ,2.Oxidative stress represents one of the main factors that negatively affect the tissue response to implantation of biomaterials, in that they are able to stimulate the formation of pro-oxidant species, contributing to the occurrence of chronic inflammation. The direct consequence is the loss of the biomaterial’s long-term biocompatibility and function because of the enrolment of leukocytes and macrophages . Thus, tThe sol–gel route has been widely employed in the preparation of biomaterials, in which organic and inorganic phases are mixed at a molecular level ,7. We ha2 flowing gas atmospheres   .In the energy-dispersive X-ray (EDX) microanalysis of the hybrids after exposure to SBF, the atomic ratio between Ca and P is equal to 1.67, depending on the number of days of exposure. While the EDX of the same samples without exposure to SBF were observed only in the peaks of the elements that formed the hybrids.Furthermore, the crystalline hydroxyapatite was confirmed by the intense peaks in the XRD spectrum . In addi· and ABTS·+ methods are useful to evaluate the radical scavenging capacity of a tested sample, providing valid, accurate and reliable data strictly related to the sample chemical features. The adopted methods, which are based on the neutralization reaction induced by the transfer of an electron (ET) and/or a hydrogen atom (HAT), are commonly used in structure–antioxidant activity relationships, which chemically depends on the presence and/or the position of active groups in investigated samples. As CGA easily reacts with both the probes in a dose-dependent manner, their use was also used to verify the CGA preservation during hybrid synthesis (supporting data acquired through characterization techniques)—or at least the maintenance of the CGA antioxidant efficacy by the conservation of those functional groups—which are commonly held responsible for the activity of the natural substance. [2/PEG50wt%/CGA5wt%), which did not show any activity towards both radicals, the other hybrids exerted a more pronounced antiradical capacity versus the radical cation ABTS. Furthermore, for each considered material, the increasing activity appeared directly correlated with the increase in the tested dose . An exception to this general trend was represented by the sample having the highest content of CGA (SiO2/PEG50wt%/CGA20wt%). The effectiveness towards ABTS+, in this case, reached a maximum—even at the 0.5 mg exposure dose—and remained constant. This showed that the effectiveness in reducing the radical probe of this hybrid is a function of the high percentage of CGA contained therein. The mechanism of chlorogenic acid in exerting free radical scavenging activity, and in particular against DPPH, has been studied by López-Giraldo et al. [The widespread and easy to use DPPHbstance. . Indeed,o et al. . The autThe MTT test was applied to evaluate if the synthesized materials had effects on cell proliferation or showed direct cytotoxic effects, thus affecting biocompatibility. This test determines the activity levels of mitochondrial dehydrogenases toward 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), which is converted to formazan crystals only by living cells . Cell viNIH-3T3 murine fibroblast, HaCat human keratinocyte and SH-SY5Y human neuroblastoma cell lines were screened. Fibroblasts and keratinocytes are suitable for the use in biocompatibility tests; fibroblasts are representative of the predominant tissue in the body and, in particular, the NIH/3T3 cell line is one of the most frequently used lines in material/cell interaction research . FurtherThese data obtained highlight that the different hybrids do not induce morphological changes and cytotoxic effects on the murine fibroblast and human keratinocyte cell lines tested. In particular, the sample containing a CGA amount equal to 15 wt% induced a slight increase in the percentage of HaCat viable cells, compared to other hybrids , whereas2/PEG/CGA hybrids—with lower amounts of the polymer—against breast cancer and osteosarcoma cell lines [On the contrary, cytotoxic effects were observed for all neuroblastoma cell samples, even when the lowest dose of chlorogenic acid (5 wt%) was entrapped. The cytotoxicity becomes more pronounced with the increase of the CGA amount, reaching about 90% of cell proliferation inhibition at the highest weight percentage . This coll lines . In thisll lines . The relll lines .The obtained results allowed us to state that synthesized materials containing different percentages of CGA determined dissimilar responses, based on the considered cell line, and in particular, hybrids are able to interfere and act selectively against tumor cells.E. coli and E. faecalis were inoculated in presence of 100 mg of the hybrid powders. E. coli was incubated with high amounts of CGA, compared with the hybrids containing only PEG. The results of the diameter of the zone of inhibition (E. coli and E. faecalis in function of the CGA content, due to disruption the bacterial cell membrane, which lead to cell death [hibition B of all ll death ,32,33. TThe damage of the bacterial cell membrane could be caused by excessive generation of ROS in the cells. In fact, it is known that the equilibrium of levels of ROS is crucial in cells. In particular, it plays many roles in intracellular signaling in different organisms, from bacteria to mammalian cells . TherefoLee et al.  suggeste2/PEG/CGA hybrids, synthesized by the sol–gel method, to neutralize free radicals, influence cell viability in vitro and inhibit bacterial growth. The radical scavenging effect seemed to be dependent on the amount of CGA and was more pronounced towards ABTS·+. The viabilities of the fibroblast and keratinocyte cell lines tested were not compromised. On the contrary, cytotoxicity of the neuroblastoma cell line was observed with increasing doses of CGA. The recorded UV–Vis spectra of all samples, compared with that of pure chlorogenic acid, suggested that there was no release of CGA, which was instead securely trapped inside the SiO2/PEG matrix. Finally, the bioactivity was not affected by the presence of CGA in the hybrids, which showed excellent antibacterial activity.The use of antioxidant biomaterials, able to control oxidant species formed in the implantation site, represents a valuable strategy to reduce all the pathological consequences derived from oxidative stress. As a part of a consolidated line of research from our group, in this work we evaluated the capability of new SiO


The serine-threonine kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast tumors, with overexpression promoting aggressive breast cancer phenotypes and poor clinical outcomes. Besides the well-defined roles of these proteins in control of cell division, proliferation, and invasion, both kinases support MAPK kinase pathway activation and can contribute to endocrine resistance by phosphorylating estrogen receptor alpha (ERα). PAK1 directly phosphorylates AURKA and its functional partners, suggesting potential value of inhibiting both kinases activity in tumors overexpressing PAK1 and/or AURKA. Here, for the first time, we evaluated the effect of combining the AURKA inhibitor alisertib and the PAK inhibitor FRAX1036 in preclinical models of breast cancer.Combination of alisertib and FRAX1036 was evaluated in a panel of 13 human breast tumor cell lines and BT474 xenograft model, with assessment of the cell cycle by FACS, and signaling changes by immunohistochemistry and Western blot. Additionally, we performed in silico analysis to identify markers of response to alisertib and FRAX1036.Pharmacological inhibition of AURKA and PAK1 synergistically decreased survival of multiple tumor cell lines, showing particular effectiveness in luminal and HER2-enriched models, and inhibited growth and ERα-driven signaling in a BT474 xenograft model. In silico analysis suggested cell lines with dependence on AURKA are most likely to be sensitive to PAK1 inhibition.Dual targeting of AURKA and PAK1 may be a promising therapeutic strategy for treatment of breast cancer, with a particular effectiveness in luminal and HER2-enriched tumor subtypes.The online version of this article (10.1007/s10549-019-05329-2) contains supplementary material, which is available to authorized users. The serine-threonine kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast tumors and associated with aggressive tumor phenotypes and poor clinical outcomes . AURKA cPAK1 gene (30% of breast carcinomas) [Increased PAK1 activity is also common in breast cancer, typically due to amplification of the cinomas) . Like AUcinomas) , 12, procinomas) , 13. PAKcinomas) . Althougcinomas) , and indcinomas) –17.Of relevance to breast cancer, both AURKA and PAK1 phosphorylate estrogen receptor alpha (ERα) (on serines S118 (PAK1) and S305 (PAK1 and AURKA)) supporting ligand-independent transcription of ERα-dependent genes promoting proliferation, invasion, and endocrine resistance , 18. TheBased on these activities of AURKA and PAK1, we hypothesized that combined inhibition of both could have synergistic anti-tumor effects in breast cancer , 21. In See “Supplementary Materials” for additional details on cell lines, cell culture, antibodies for Western blot and IHC, drug formulations for xenograft experiments, and statistical analysis.Human breast cancer cell lines from the American Type Culture Collection were cultured in standard conditions. We confirmed negative mycoplasma testing and STR profile for each cell line. Alisertib was purchased from MedChem Express . FRAX1036 was synthesized by AK and WW .Cells were grown on 96-well plates for 24 h before treatment with drug(s) or vehicle. Cell viability was measured by CellTiterGlo assay  after 72 h of treatment. Each drug concentration was evaluated in triplicate, with ≥ 3 biological repetitions. We determined synergy by Chou-Talalay method .t test and one-way ANOVA.Protein lysates were prepared with RIPA lysis buffer  containing protease/phosphatase inhibitor . Each blot was repeated with ≥ 3 preparations of lysates. Signal intensity was quantified by NIH ImageJ Software, or Odyssey imager software , normalized to vinculin or GAPDH, and compared by two-tailed scid Il2rgtm1Wjl/SzJ (NSG) mice from the FCCC breeding colony were maintained under pathogen-free conditions. Estrogen pellets were implanted subcutaneously into 6- to 8-week-old mice as described [7 BT474 cells (N = 45 mice). Treatment consisted of alisertib (15 mg/kg twice a day), FRAX1036 (20 mg/kg daily) or combination of drugs; control group received vehicle solution twice a day; all agents were administered by oral gavage.All animal experiments were approved by the FCCC Institutional Animal Care and Use Committee. NOD.Cg-Prkdcescribed ; simulta3, mice were treated for 3 days with vehicle, alisertib, FRAX1036, or combination of drugs, then euthanized and tumors were frozen for Western blots. To assess long-term responses, once tumors reached 150 mm3, mice were treated for 21 days, then euthanized, and tumors collected for analysis.To assess short-term signaling, after tumor volume reached 600 mmIHC was performed according to standard protocols. Results were quantitated with Aperio ePathology  and analyzed by Mann–Whitney and Kruskal–Wallis tests.Non-synchronized growing cells were fixed with ethanol at 24 and 72 h after treatment with drug(s) or vehicle, then mixed with propidium iodide solution  before FACS ; data were analyzed by one-way ANOVA.z-score normalized Gene Activity Ranking Profile (zGARP) score have been described in detail [AURKA, CCND1, MYC, PAK1-3, and TFF1 were extracted from [PAK1-3, we selected the smallest of the zGARP scores for each cell line. RNAseq fragments per kilobase million (FPKM) values were extracted from [p values were calculated using GraphPad Prism for the drug IC50 versus zGARP score.Methods for deriving n detail , 26. zGAted from . For PAKted from , 27–29. We evaluated the effect of dual inhibition of AURKA and PAK1 on the proliferation of 5 luminal , 4 hormone receptor negative (HR-) human epidermal growth factor receptor 2 positive (HER2 +) , and 4 triple negative (TNBC)  breast cancer cell lines  and BT474 (HR +/HER2 +) cell lines for evaluation of cell cycle and signaling changes upon co-inhibition  and ERα(S305) in both cell lines Fig. e, f. AliWe evaluated the drug combination in vivo using BT474 (HR +/HER2 +) orthotopic xenografts. Tumors were established in NOD/SCID mice and treated for 21 days with vehicle, FRAX1036 20 mg/kg, alisertib 15 mg/kg, or combined FRAX1036/alisertib , but not in FRAX1036-treated mice . Although FRAX1036 produced initial responses, they were lost after 10 days ; further, tumor volume with the combination treatment was smaller comparing to monagent alisertib (p = 0.004). Importantly, only the alisertib/FRAX1036 combination reduced tumor volume compared to the initial volume (~ 150 mm3)  and one case of complete response in treated mice. All therapies were well tolerated, with weight of drug- and vehicle-treated mice not significantly differing  and treated mice with vehicle, alisertib, FRAX1036, or the combination for 3 days, then analyzed tumor lysates. FRAX1036 effectively reduced levels of phospho-PAK1/2/3 , C-MYC, and TFF1. Integration of four RNAseq datasets confirmed the expected higher expression of C-MYC and TFF1 in ERα + versus ERα- subsets  score [AURKA, PAK1-3, CCND1, C-MYC, and TFF1 with response to alisertib and FRAX1036 in our cell line experiments  score , 26. TheP) score . We corrOur results indicate that combined inhibition of AURKA and PAK1 is of potential value for the treatment of breast cancer, with greatest efficacy seen in luminal HR + and HER2 + subtypes in vitro. This could be explained by the interaction of AURKA and PAK1 with ERα (phosphorylation leading to ligand-independent activation), and with HER2 , 18. AURThe potency of the combination in luminal cell lines is likely due at least in part to the decreased phosphorylation of ERα at both the S305 and S118 residues, seen both in vitro and in xenograft experiments. Greater disruption of cell cycle control with the combination is also likely to contribute. In the BT474 xenograft model, the combination effectively inhibited signaling proteins linked to G1 and G2/M cell cycle control and ERα-activation, including cyclin B1, TFF1, C-MYC, and cyclin D1. This was consistent with the FACS analysis showing the combination arrested BT474 cells in both G1 and G2/M phases. One limitation of the present work is that we did not use cell sorting to separate mouse stromal cells from human breast cancer cells in these experiments; this may have led to somewhat diminished apparent effect of the drugs on phosphorylation of ERα.AURKA in ERα + tumors, providing a rationale to combine AURKA and PAK1-inhibitors.We have expected a synergistic effect of alisertib and FRAX1036 on cell cycle and suppression of tumor growth because of more effective suppression of AURKA in the settings of PAK1 inhibition . HoweverC-MYC promoter, enhancing breast cancer stem cell phenotypes [C-MYC knock down required higher concentrations of alisertib for growth inhibition. While cell lines highly dependent on C-MYC have more compensatory mechanisms to escape alisertib-induced C-MYC downregulation, co-treatment with PAK1 inhibitors may abrogate these mechanisms, allowing response to lower doses of alisertib.The combination effectively inhibited expression of the transcription factor and proto-oncogene C-MYC, a protein frequently overexpressed in breast tumors, and implicated in poor clinical outcomes , 40. Desenotypes . In paraenotypes , 45. SigTogether, our results provide evidence that dual inhibition of AURKA and PAK1 is of value in breast cancer. Enhanced anti-tumor activity of this combination is based on multiple mechanisms, including enhanced inhibition of phosphorylation of AURKA, PAK1, and ERα, as well as decreased expression of cell cycle proteins and C-MYC. Although resistance developed in vivo to single agent FRAX1036, addition of FRAX1036 to alisertib conferred significant advantages and lead to cases of complete or near complete tumor response, consistent with the concept that combination targeted therapy is beneficial because of synergistic anti-tumor effect and prevention of the selection of drug-resistant subclones during therapy .One limitation of our study is that we examined the effects of the combination in a single in vivo model—further studies in PDXs and breast tumor cell organoids will be useful to confirm and extend our findings. In our study, as proof of concept, we used a prototype PAK1/2/3 inhibitor FRAX1036. Newer, more potent and selective PAK1 inhibitors now in development , 48 shouSupplementary material 1 (DOCX 26 kb)Supplementary material 2 (PPTX 8588 kb)Below is the link to the electronic supplementary material.


Triterpenoids are the most abundant secondary metabolites present in marine organisms, such as marine sponges, sea cucumbers, marine algae and marine-derived fungi. A large number of triterpenoids are known to exhibit cytotoxicity against a variety of tumor cells, as well as anticancer efficacy in preclinical animal models. In this review efforts have been taken to review the structural features and the potential use of triterpenoids of marine origin to be used in the pharmaceutical industry as potential anti-cancer drug leads. Geodititoxicity .35–38), and erylosides F (39) . Four compounds induced the early apoptosis of Ehrlich carcinoma cells, where erylosides F3 have shown the highest activity at a concentration of 100 µg/mL [Five cytotoxic triterpene glycosides, erylosides F1-F4 (s F (39)  were iso00 µg/mL .40–48) and sodwanones K-W (49–61), have been isolated from the Indo-Pacific sponge Axinella wltneri [46), H (47), and I (48) have been found to have cytotoxic activity. The compounds have shown cytotoxicity activity against cell cultures of P-388 murine leukemia, A-549 human lung carcinoma, HT-29 human colon carcinoma, and MEL-28 human melanoma. Sodwanones G (46), H (47), I (48) showed high specificity towards human lung carcinoma cell line A-549, where the specificity of sodwanone G was prominent (46) [49), L (50), and M (51) were found to be cytotoxic to P-388 murine leukemia cells [57) was evaluated against 13 human tumor cell lines [60) inhibited both hypoxia-induced and iron chelator -induced HIF-1 activation in T47D breast tumor cells (IC50 15 µM), and sodwanone V (60) was the only sodwanone that inhibited HIF-1 activation in PC-3 prostate tumor cells (IC50 15 µM).The special group of triterpenoids named sodwanones: sodwanones A-I ( wltneri . Sodwanoent (46) . The cytia cells . The bioll lines . Sodwano40) and sodwanone T (58) inhibited hypoxia-induced HIF-1 activation in T47D cells , and sodwanone V (60) showed cytotoxicity to MDA-MB-231 breast tumor cells (IC50 23 µM). Sodwanone derived compounds, 3-epi-sodwanone K (62), 3-epi-sodwanone K 3-acetate (63), 10,11-dihydrosodwanone B (64) have been isolated from Axinella sp., and 62 and 64 also inhibited hypoxia-induced HIF-1 activation in T47D cells  and 63 was cytotoxic to T47D cells (IC50 22 µM) [Sodwanone A (0 22 µM)  Figure 0 and sod65–83 (65), raspacionins A (66), raspacionins B (67), 21-deacetylraspacionin (68), 10-acetoxy-21-deacetyl-28-hydroraspacionin (69), 10-acetoxy-21-deacetyl-4-oxo-28-hydroraspacionin (70), 10-acetoxy-15,21-dideacetyl-4-oxo-28-hydroraspacionin (71), 10-acetoxy-15-deacetyl-4-oxo-28-hydroraspacionin (72), 10-acetoxy-4-acetyl-15-deacetyl-28-hydroraspacionin (73), 10-acetoxy-15-deacetyl-4-21-dioxo-28-hydroraspacionin (74), 10-hydroxy-4,21-dioxo-28-hydroraspacionin (75), 21-oxoraspacionin (76), 15-deacetyl-21-dioxo-raspacionin (77), 4,21-dioxo-raspacionin (78), 10-acetoxy-4,21-dioxo-28-hydroraspacionin (79), 10-acetoxy-4-acetyl-21-oxo-28-hydroraspacionin (80), 10-acetoxy-4-acetyl-28-hydroraspacionin (81), 10-acetoxy-28-hydroraspacionin (82) and 10-acetoxy-21-deacetyl-4-acetyl-28-hydroraspacionin (83), have been isolated from red sponge, Raspaciona aculeuta Johnston (family Raspailiidae), and from the Mediterranean sponge Raspaciona aculeata. All the compounds have showed cytotoxicity against MCF-7 tumor cell line with IC50 values between 4 and 8 µM [Raspacionin triterpinoids 65–83 , namely and 8 µM ,35,36.Siphonochalina siphonella is a rich source of sipholane triterpenoids including sipholenols  , sipholenones  , and siphonellinols  . Sipholenol A (84) and sipholenone A (sipholenol B) are the major sipholane triterpenoids [84), sipholenol I (91), sipholenol L (94), sipholenone A (95), sipholenone E (96), siphonellinol C (97), and siphonellinol D (98) have found to show potent reversal of multidrug resistance in cancer cells that over expressed P-glycoprotein. These compounds enhanced the cytotoxicity of several P-glycoprotein substrate anticancer drugs, and significantly reversed the multidrug resistance phenotype in P-glycoprotein-overexpressing multidrug resistant cancer cells KB-C2 and KB-V1 in a dose-dependent manner [The Red Sea sponge rpenoids . Sipholerpenoids . Sipholet manner ,40 , B (101), and C (102), pervicoside C (103) and holothurin A (104) isolated from Holothuria fuscocinerea Jaeger on human leukemia HL-60 and human hepatoma BEL-7402 cells was analyzed and all compounds have shown a potent cytotoxicity towards both cell lines. However, fuscocineroside C was found to be the most potent  in HL-60 and BEL-7402 cell lines respectively [Triterpenoid glycosides (saponins) are the major and most abundant type of compounds isolated from sea cucumbers. Saponins are generally perceived as highly active natural product and the sea cucumber saponins have been well characterized for their anti-cancer activities. The cytotoxicity of five triterpene glycosides, fuscocineroside A  and A4 (106) found to be strongly cytotoxic to cancer cell lines; human epidermoid carcinoma (KB) and human hepatocellular carcinoma (Hep-G2), with IC50 values of 0.87 and 0.32 µg/mL (for compound 7) and of 1.12 and 0.57 µg/mL (for compound 8), respectively [The triterpene glycosides from the sea cucumber ectively   also exhibited significant cytotoxicity against different human tumor cell lines while showing the highest activity towards human colorectal carcinoma (HCT-116) cells (IC50 = 0.14 µM) with more potency than the employed positive control, 10-hydroxycamptothecin (IC50 = 0.84 µM) [108) and C (109) have also shown potent cytotoxicity against human tumor cell lines, adenocarcinomic human alveolar basal epithelial cells (A549), HCT-116, HepG2, and human breast adenocarcinoma (MCF-7) cell lines. The cytotoxicity of the compounds on A549  and HCT-116  cells were more potent than the positive control V-16 (110) and E (111) have also been tested for their anticancer activities in above human cancer cell lines and revealed a significant activity with IC50 values in the range of 3.36–7.77 µg/mL [108 and 109 are potent cytotoxic agents compared to compounds 110 and 111. It has been reported that the length and type of sugar moieties of glycosides play an important role in terms of cytotoxic activity against tumor cells and this observation clearly indicates that.Arguside A (0.84 µM) . Argusidrol V-16 . However77 µg/mL  Figure 7 also exin vitro cytotoxicity of impatienside A (112) and bivittoside D (113) were evaluated extensively by employing seven human cancer cell lines and the results showed that both glycosides exhibited in vitro cytotoxicities similar to or better than that of the potent anticancer drug etoposide (V-16) in four human tumor cells, A549 , HCT-116 , DU-145 , KB . The structural differences between glycosides 112 and 113 limited to their holostane skeleton, and no significant difference in the cytotoxicity of the two glycosides was found. However, pervicoside C (103), an analogue of 113 having the same aglycone but a different sugar chain, isolated from Holothuria fuscocinerea Jaeger, exhibited weak activities against HCT-116 and A549 cancer cells, with IC50 values of 18.7 and 28.6 µg/mL, respectively [Moreover, the ectively . Accordi114) and griseaside A (115) are identified as promising anticancer agents due to their significantly higher cytotoxicity against four human tumor cell lines, A549  , HL-60 , BEL-7402 , and human acute lymphoblastic leukemia cell line (Molt-4)  compared to the positive control HCP  [17-Dehydroxyholothurinoside A ( 2.2 µM)  Figure 4 and gri116) has also been tested for its anticancer potential against eight human tumor cell lines  and has exhibited cytotoxicity with IC50 values in the range of 0.15–3.20 µg/mL [116 has shown more potent cytotoxicity towards CAKI-1 (IC50 = 0.15 µg/mL) and KB-VIN (IC50 = 2.81 µg/mL) cell lines. Three new triterpene glycosides, intercedensides A (117), B (118), and C (119) from Mensamaria intercedens Lampert, were widely studied for their anticancer activity employing 10 human tumor cell lines . Interestingly all compounds showed a significant cytotoxicity against all tumor cell lines within the IC50 value range of 0.7–4 µg/mL, and the compounds 117 and 119 showed similar potencies, while compound 118 was generally more potent in all cell lines. Furthermore, compound 117 also exhibited significant in vivo antineoplastic activity against mouse Lewis lung cancer and mouse S180 sarcoma, with 48.39% and 57.48% tumor reduction levels [Hillaside C  showed a significant cytotoxicity (IC50 = 0.75–3.50 µg/mL) against ten tumor cell lines  [121) from Pseudocolochirus violaceus exhibited significant cytotoxicity against cancer cell lines MKN-45 (human gastric adenocarcinoma) and HCT-116 with IC50 values in the range of 0.052–0.442 µM and both compounds showed significantly higher activity against HCT-116 compared to the positive control HCP [A new sulfated triterpene glycoside from ls-A431) . Further122) and B (123) showed significant cytotoxicity (IC50 = 0.75–3.50 µg/mL) against ten tumor cell lines  [Moreover, the sulfated triterpene glycosides, philinopsides A (and PC3)  , cucumarioside A2-2 (125), echinoside A (126) and ds-echinoside A (127) have been reported against several cancer types in vitro and in vivo (in vivo studies have confirmed that frondoside A (100 µg/kg/day) effectively decreased the growth of breast cancer xenografts in athymic mice without exerting any side-effects [−1) to the mice bearing H22 hepatocarcinoma tumors has reduced the tumor weight by 49.8% and 55% respectively [Even though a number of saponin compounds have been isolated and identified as potent cytotoxic agents only few of them have been studied to unravel the mode of their cytotoxicity. Among them detailed cytotoxic mechanisms of frondoside A ( in vivo . All fou-effects . Moreoveectively . These s128) and (21a)-21-hydroxythyrsiferol (129) were isolated and identified from the marine red alga Laurencia mariannensis, which was collected off the coast of Hainan and Weizhou Islands of China. Both compounds have displayed significant cytotoxic activity against P-388 tumor cells with IC50 values of 0.6 and 6.6 mg/mL, respectively [Two cytotoxic squalenoid-derived triterpenoids, laurenmariannol , isodehydrothyrsiferol (131) and 10-epidehydrothyrisiferol (132), isolated from Laurencia viridis, have shown potent cytotoxic activities against a number of cancer cell lines [The red seaweed ll lines ,57.133), 22-hydroxy-15(28)-dehydrovenustatriol (134), 1,2-dehydropseudo-dehydrothyrsiferol (135), and secodehydrothyrsiferol (136) exhibited significant cytotoxic activity against a panel of cancer cell lines [137), thyresenol A (138) and thyrsenol B (139) were also isolated from Laurencia viridis, and these compounds have exhibited significant inhibitory action on protein phosphatase at a concentration of 10 mM. Moreover, they have shown potent cytotoxic activity against P388 cell line [Polyethers, iubol , l5-anhy-drothyrsiferyl diacetate diacetate (141), magireol-A (142), magireol B (143) and magireol C (144) were isolated from Japanese red alga Laurencia obtuse [Five cytotoxic triterpenoids 28-anhy-drothyrsiferyl diacetate , and cyclosadol (146) were isolated from brown algae Kjellmaniella crassifolia, two compounds were obtained from this species for the first time. 3-epicyclomusalenol (145), and cyclosadol (146) have been reported to have moderate chemo preventive effects [Two cycloartane-type triterpenoids, 3-epicyclomusalenol  were isolated from the fermented extract of Xylarialean sp. A45, an endophytic fungus of Annona squamosa L., and their structures were determined by NMR spectroscopy. These compounds have shown modest cytotoxic activities against human tumor cell line HepG2 [Triterpenoids are frequently found in marine source, but have rarely been reported from marine-derived fungi. Rainer Ebel have reviewed 7 triterpenoids from marine-derived fungi . In 2011ne HepG2  (Figure et al. [Even though not much research in this area has been carried out, the anti-cancer activity of triterpenes is believed to be directly correlated to their structural features. As suggested by many authors the bioactivity of the triterpenes is a result of its strong membranolytic activity, and this membranolytic activity is a function of the structural features of the glycosides . In tritet al.  have repet al. .Up to date the vast chemodiversity in the oceans has paved the way for natural product chemists to mine for new bioactive compounds. Among them triterpenoids are one of the most studied classes of compounds. Due to the extreme environments in the oceans, survival demands have resulted in the evolution of these sophisticated toxic compounds and this fact is confirmed by the proven toxicity of these compounds in biochemical studies. Triterpenoids derived from sea cucumbers, sponges and algae have been used as ingredients in Traditional Chinese Medicine for years. Even though there are many lead compounds with promising potential to be used as drugs for cancer therapy, the cytotoxicity itself would be a constraint for this purpose, because most of the compounds could be cytotoxic towards normal cells in addition to the cancerous cells. In identification of therapeutics from natural products the preference is given to the compounds having high specificity towards the cancer cells in their cytotoxic action, while minimizing the damage to normal cells . TherefoMoreover, the possibility of continuous supply of the product and the ecological importance of the triterpene sources such as sponges, sea cucumbers and algae are factors of importance before entering to the drug development phase. Sustainable production of compound through chemical synthesis or culturing of these marine organisms should be ensured. The structural complexities have challenged the chemical synthesis and thus it would limit the entering of these compounds to drug development phase. However, with the advances in synthetic chemistry and understanding of triterpene biosynthetic processes, new opportunities for exploitation of these compounds as drug leads are opening up.