Background: To test and introduce effective and less toxic breast cancer (BC) treatment strategies, animal models, including murine BC cell lines, are considered as perfect platforms. Strikingly, the knowledge on the genetic background of applied BC cell lines is often sparse though urgently necessary for their targeted and really justified application. Methods: In this study, we performed the first molecular cytogenetic characterization for three murine BC cell lines C-127I, EMT6/P and TA3 Hauschka. Besides fluorescence in situ hybridization-banding, array comparative genomic hybridization was also applied. Thus, overall, an in silico translation for the detected imbalances and chromosomal break events in the murine cell lines to the corresponding homologous imbalances in humans could be provided. The latter enabled a comparison of the murine cell line with human BC cytogenomics. Results: All three BC cell lines showed a rearranged karyotype at different stages of complexity, which can be interpreted carefully as reflectance of more or less advanced tumor stages. Conclusions: Accordingly, the C-127I cell line would represent the late stage BC while the cell lines EMT6/P and TA3 Hauschka would be models for the premalignant or early BC stage and an early or benign BC, respectively. With this cytogenomic information provided, these cell lines now can be applied really adequately in future research studies.  Breast cancer (BC) is among the most common female specific cancer types and the second most common cancer in humans after lung cancer ,2. SurviThere are five stages of BC: (i) benign, premalignant stage; (ii) atypical ductal hyperplasia; (ii) preinvasive stage of ductal carcinoma in situ; (vi) metastatic carcinoma; and (v) advanced stage . Known gBC subtyping is extremely important for metastasis staging and treatment ,10,12,13However, the need for new types of medication with less side effects and being best targeted is still high . TherefoFluorescence in situ hybridization (FISH) is considered the most practicable technique to detect gross genetic alteration in cancer . Thus, iAs previously done in comparable studies in murine tumor cell lines, a successful in silico translation from murine to human genome determined the corresponding homologous genetic alteration in human BC and enabled a classification as murine late stage, premalignant stage and benign BC-models.Murine BC cell line C-127I presented as a pentaploid but was basically genetically relatively instable and was rearranged with more than 10 derivative chromosomes. Consequently, this cell line can be divided to four clones, clone 1 being the ancestor clone and clones 2, 3 and 4 being derivatives of that. Clone 1 was present in 20% of the cells and can be described as 94~96,XXX,-2,der(4)t,der(4)t,+der(4)t,der(6)(pter→A1::G3→E::B3→D:),del(6)(D),+del(6)(A2),−7,+8,−9,dic,inv(12)(BE),−12,−12,del(13)(B1),del(14)(D3),−14,+15,−16,+17,−18,−18,+19.Clone 2, being present in 40% of the cells had the same karyotype as clone 1 with two additional aberrations, i.e., loss of one chromosome 8 and +der(17)t instead of a normal chromosome 17, as shown in For clone 3, representing 23% of the cells compared to clone 1, one chromosome 12 was lost and one was replaced by a derivative chromosome 12: del(12)(A1.1).Clone 4 formed the remaining 17% of cells—here, an additional der(5)t and a idic(18) were present as a structural aberrations and one chromosome 10 and 18 each were lost.This cell line EMT6/P is triploid and can be divided into five main clones which show some chromosomal instability. Clone 2 can possibly be considered the “ancestor” clone.The largest clone (clone 1) represents 40% of the cells, with the following karyotype : 59~64,XIn clone 2 (20%), the breakpoints of der(5)t were different than in clone 1 — here, they were in 5B1 and 15D1 as der(5)t compared to a der(5)(5A1→5C3::5B1→5C3::15D1→15qter) in clone 1.Clone 3 (10%) compared to clone 1 had no normal chromosome 12 and instead a second idic(12)(A1A1). Clone 4 (7%) had no additional chromosome 3 and a complex reciprocal translocation t with a der(3)t(3pter→3H1::2?→2?qter) and der(2)t(2?pter→2?::3H1→3F2:): the clone could not be found in mcb2 analyses; thus, breakpoints for chromosome 2 could not be determined. Clone 5 (23%) showed reciprocal translocations as follows: t and t; moreover, +der (9) t(9pter→9?::18A2→18qter) and +der(18)t(18pter→18E2::9?→9qter) were present compared to clone 1.This cell line TA3 Hauschka had a particularly stable diploid karyotype and can be divided in three clones for which probably clone 1 was an ancestor of clones 2 and 3. Kayrotype of clone 1 is shown in 41,X,der(X)(XA1→XA5::19C2→19D2::19D1→19qter),t,t,inv(3)(E1H4),del(4)(C3D2),t,+6,t,+16,der(19)t,del(19)(D2).Clone 2 (30%) was characterized by an additional reciprocal translocation t and a more complex derivative of chromosome 19, der(19)(19pter→19C2::XA6→XF5::8D1→8qter), compared to clone 1. Clone 3 (20%) differed from clone 1 by a der(4)t instead of del(4)(C3D2).The aforementioned FISH studies of the three murine BC cell lines were in line with the aCGH results and are summarized in The three studied BC cell lines present acquired copy number variations in regions known to harbor oncogenes and tumor suppressor genes, related to human BC ,26; as sBC has several subtypes  due to tHER2-enriched subtype which is known as an aggressive subtype and resistant to treatment [The three cell lines showed differences in the genomic alteration rate regarding ploidy, numerical and structural aberrations, and tumor-associated breakpoints. C-127I presented a very complex karyotype with pentaploidy, EMT6/P just was triploid and had less structural aberrations than C-127I, while TA3-Hauschka was near diploid and had only few imbalances. Thus, this is the first evidence that the three cell lines may demonstrate different subtypes and stages of human BC . Howeverreatment ,34. As all three cell lines were established between 34 and 67 years ago ,23,24, terbb2 gene is localized, while gain in the brca1 region was only evident for C-127I. Also, oncogene myc, being amplified in many human tumors, showed gain of copy numbers in C-127I and EMT6/P but not in TA3 Hauschka. Tumor suppressor gene rb1 was only deleted in C-127I. As highlighted in p53 pathway regulation, is affected by chromosomal break events in all three studied murine cell lines. The aforementioned pathways are affected in aggressive forms of BC [DICER1 is located, recently identified as playing a role in cancer predisposition [As according to ploidy, karyotype and CNVs, C-127I is the most aberrant cell line, followed by EMT6/P and TA3 Hauschka; the same tendency is emphasized when looking at the number of chromosomal breakpoints and their localizations . The murms of BC ,46. The ms of BC ,31,47. Fms of BC . Moreoveposition ,32. Horlings and coworkers correlated in 2010  the freqESR1; in human 6q25.1). However, there was a loss in gene estrogen receptor 2  in C-127I. The latter cell line presents also gains in progesterone receptor gene . Finally, one may speculate about the influence of breakpoints and/or CNVs of regions being involved in presenting ICM, like ER or PR; in none of the three cell lines, there are changes in regions being homologous to gene estrogen receptor 1  was purchased from CLS Cell Lines Service GmbH , while the EMT6/P-  and TA3 Hauschka-cell line  were obtained from European Collection of Authenticated Cell Cultures . They were grown adherently in DMEM medium (C-127I) or in EMEM/EBSS medium (EMT6/P and TA3 Hauschka) according to company instructions. Cells were prepared cytogenetically as previously reported ; whole gFISH was performed as previously described . In shorThe breakpoints and imbalances of the three studied BC cell lines were determined according to aCGH and mcb data and aligned to human homologous regions using Ensembl Browser, as previously described . The obt


However, the process carried out to obtain immortalized cell lines could profoundly alter gene expression profile, and consequently their biological characteristics, leading to debatable results. Here, we evaluated the still undisclosed similarities and differences between HS-5, HS-27A cell lines and primary bone marrow MSCs in the context of tumor development and immunomodulation. Furthermore, we assessed by standardized immunological assays the capability of the cell lines to reproduce the general mechanisms of MSC immunoregulation. We found that only HS-5 cell line could be suitable to reproduce not only the MSC capacity to influence tumor biology, but also to evaluate the molecular mechanisms underlying tumor immune escape mediated by stroma cells. However, HS-5 pre-treatment with inflammatory cytokines, that normally enhances the immunosuppressive activity of primary MSCs, did not reproduce the same MSCs behavior, highlighting the necessity to accurately set up in vitro assays when HS-5 cell line is used instead of its primary counterpart.In this study, we compared the overall gene and pathway expression profiles of HS-5 and HS-27A stromal cell lines with those of primary bone marrow MSCs to verify if they can be considered a reliable alternative tool for evaluating the contribution of MSCs in tumor development and immunomodulation. Indeed, due to their easier manipulation  In addiipocytes . MSC-medipocytes . The welipocytes . Moreoveipocytes .In the last years, MSCs have been further recognized as crucial facilitators of tumor development in the context of both solid and liquid cancers. Emerging data suggest that MSCs can promote different tumor processes, including malignant transformation, angiogenesis, metastasis formation, cancer cell survival and chemoresistance . Last, bin vitro, with low variability of the results obtained in presence of stromal cells. Therefore, several research groups take advantage of such commercially available cell lines to study the mechanisms mediated by MSCs in influencing immune responses and tumor progression  database was searched for datasets with publicly accessible datasets with the keywords MSC, HS-5 and HS-27A. We selected four microarray datasets, GSE9593 , GSE1059 package ; multipl package . Gene se package  and to o package  and the ® . Both primary MSCs and cell lines were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2% L-Glutamine . All experiments were performed between passages 2 and 7 of primary MSCs. Cells at 80% confluence were treated or not for 48 h with 10 ng/mL IFN-γ and 15 ng/mL TNF-α (R&D Systems) to induce inflammatory priming. PBMCs were isolated from human blood using Lymphoprep (Stem cells Technologies). B, T, and NK lymphocytes were isolated from PBMCs using immunomagnetic negative selection (Miltenyi Biotec) with at least 95% cell purity, as evaluated by flow cytometry. PBMCs were stimulated with 5 μg/ml of phytohemagglutinin (PHA) (Sigma-Aldrich) for 4 days in IMDM supplemented with 10% pooled human AB serum, 1% penicillin-streptomycin and 2% L-Glutamine . T cells were activated with 0.5 μg/mL cross-linking anti-CD3 and anti-CD28 antibodies (Sanquin) for 6 days in RPMI supplemented with 10% human AB serum, 1% penicillin-streptomycin and 2% L-Glutamine . B cells were activated with 5 μg/mL antihuman IgM+IgA+IgG (F(ab’)2, Jackson Immunoresearch), 50 IU/mL rhIL-2 (Novartis), 50 ng/mL polyhistidine-tagged CD40 ligand, 5 μg/mL anti-polyhistidine antibody (R&D Systems), and 0.5 μg/mL CpG ODNs (Invitrogen), in RPMI medium supplemented with 10% FBS, 1% penicillin-streptomycin and 2% L-Glutamine . The identity of HS-5 and HS-27A was checked for the presence of mesenchymal markers. Cell suspension were stained using the antibody anti-human CD73-PE, CD90-PE, CD105-PE, CD14-PE, CD31-PE, CD34-PE, and CD45-PE, HLA-ABC-PE, HLA-DR-PE, Fas-FITC, FasL-PE (BD Bioscience). The inflammatory immunophenotype was established using the anti-human CD54-PE, CD106 PE, HLA-ABC-PE, HLA-DR-PE, CD274-PE monoclonal antibodies (BD Bioscience). Peripheral blood mononuclear cells (PBMCs) were characterized using the anti-human CD3-FITC, CD16/56-PE, CD45-PerCP, CD19-APC, CD4-APC-H7, and CD8-PECy7 monoclonal antibodies (BD Bioscience). All data were collected through flow cytometry  and analyzed with FlowJo software (TreeStar). The expression of MSC markers was analyzed on living cells by using TO-PROTM-3 Iodide (Thermo Fisher) and normalized on FMO (fluorescence minus one) control. Osteogenic and adipogenic differentiative ability of primary MSC and γ-irradiated HS-5 and HS-27A cells (20 Gy – 137Cs as source of γ-radiation) was evaluated has already described  and characterized as already described . HS-5 anescribed . Primary5 PBMCs, 2 × 105 T cells, 2 × 104 B cells, or 2 × 104 NK cells were added. At the end of the co-culture, cells were harvested at the stained with mouse anti-human CD45-PerCP-Vio700 (Miltenyi Biotec), and TOPRO-3 Iodide (Life Technologies). The proliferation was assessed on viable TOPRO-3 negative and CD45 positive cells by FlowJo software (TreeStar) by using the CFSE Geometric Mean of proliferating cells. The same experimental procedures were carried out using γ-irradiated HS-5 and HS-27A cells (20 Gy – 137Cs as source of γ-radiation).Standardized assays were carried out to assess the inhibitory functions of primary MSCs and cell lines on different IECs, as previously described by our group . Either According to the minimal criteria for defining MSCs established by the International Society for Cellular and Gene Therapy (ISCT), primary bone marrow MSCs and HS-5 and HS-27A cell lines were positive for CD73, CD90, CD105, and HLA-ABC, with no expression of CD14, CD31, CD34, CD45, and HLA-DR surface molecules . However2. For the initial evaluation of potential differences or similarities between primary MSCs and cell lines, we considered two general MSigDB gene set collections covering a good portion of the human cellular and biological pathways, i.e., C2 and C5 collections. C2 collection includes several gene sets deriving from various sources, i.e., online pathway databases and the biomedical literature. The C2 collection is divided into two sub-collections: Chemical and genetic perturbations (CGP) and Canonical pathways (CP). The majority of the CGP sets came from the biomedical literature, thus identifying different signatures of biological and clinical states, such as cancer metastasis, stem cell characteristics, etc. The CP sub-collection includes several pathway gene sets from commonly used online databases, including BioCarta, KEGG, Matrisome Project and others. C5 collection consists of gene sets derived from Gene Ontology (GO) annotations. Therefore, the C5 collection is based on GO terms, belonging to the three GO ontologies , and their associations to human genes. Considering both C2 and C5 collection, the PCA analysis on differentially expressed pathways (DEP) clearly showed two distinct clusters. HS-5 cell line and primary MSCs clustered together, whereas HS-27A cell line represented a distinct group , CD106 (V-CAM), HLA-ABC, and HLA-DR (MHC-II), and CD274 (PD-L1) . FurtherWe further investigated HS-5 cell line immunological properties towards purified T, B, and NK cells by using standardized immunological assays. As already published by our group , restingin vitro and in vivo  signaling pathways” is concerned, two pathways strictly related to MSC immune regulatory effect. All these data were then confirmed by the functional assays we performed on activated PBMCs and purified IECs, although HS-5 pre-treatment with inflammatory cytokines, that normally enhances the immunosuppressive activity of primary MSCs, did not affect PBMC proliferation. This difference requires further investigation, because it could reflect either a different sensitivity of HS-5 cell line to inflammatory priming or a persistent status of intracellular activation.in vitro as compared to primary MSC cultures. However, we strongly highlight and recommended to accurately set up the immunological assays when HS-5 cell line is used instead of its primary counterpart.Taken together, these data indicate that HS-5 cell line is suitable to reproduce not only the MSC capacity to influence tumor biology, but also to evaluate the molecular mechanisms underlying tumor immune escape mediated by stroma cells, with a number of advantages due to its easier manipulation + cells isolated from myelodysplastic syndrome patients to promote the engraftment of clonal hematopoietic precursor. Furthermore, human CD34+ precursors harvested from bone marrow and spleen of primary murine recipients, when combined with HS-27A cells, were also engrafted successfully in secondary NSG recipients, showing the persistence of the original clonal characteristics (The pronounced differences between HS-5 and HS-27A reported in this work is further supported by eristics . The auteristics . TherefoThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethical Committee of Azienda Ospedaliera Universitaria Integrata Verona; N. 1828, May 12, 2010 “Institution of cell and tissue collection for biomedical research in Onco-Hematology”. The patients/participants provided their written informed consent to participate in this study.AA designed, performed the laboratory work, and wrote the manuscript. PD performed statistical and gene expression profile analyses and wrote the manuscript. AG, AB, AM, PT, RB, and SC performed the laboratory work and contributed to manuscript writing. SU critically revised the manuscript. MK coordinated the research plan, wrote and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although several antipsychotic drugs have been shown to possess anticancer activities, haloperidol, a “first-generation” antipsychotic drug, has not been extensively evaluated for potential antineoplastic properties. The aim of this study was to investigate the antitumoral effects of haloperidol in glioblastoma (GBM) U87, U251 and T98 cell lines, and the effects of combined treatment with temozolomide (TMZ) and/or radiotherapy, using 4 Gy of irradiation. The viability and proliferation of the cells were evaluated with trypan blue exclusion assay and 3--2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis, using the annexin-propidium iodide (PI), and cell cycle, cluster of differentiation (CD) expression and caspase-8 activation were measured using flow cytometry. Treatment with haloperidol significantly reduced cell viability in U87, U251 and T98 GBM cell lines. Haloperidol induced apoptosis in a dose-dependent manner, inhibited cell migration and produced an alteration in the expression of CD24/CD44. The additional effect of haloperidol, combined with temozolomide and radiation therapy, increased tumor cell death. Haloperidol was observed to induce apoptosis and to increase caspase-8 activation. In conclusion, haloperidol may represent an innovative strategy for the treatment of GBM and further studies are warranted in glioma xenograft models and other malignancies. Glioblastoma (GBM) is the most aggressive glial brain tumor in adults, with a median life expectancy of 15 months, despite gross total excision, radiotherapy and temozolomide (TMZ)-based chemotherapy . In addi2 at 37 °C. Before every experiment, haloperidol  and TMZ  were diluted in dimethyl sulfoxide (DMSO) from stock solution (1 mM) to the final concentration with culture medium. Cultures of malignant glioma cells were treated with haloperidol or TMZ, or both, with and without radiotherapy.The human glioma cell lines U87 and U251 were obtained from Dr W.K. Alfred Yung  and T98 were obtained from ATCC . All cell lines were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco BRL). All cell lines were incubated in a humidified atmosphere containing 5% COFor the viability assays, glioma cell lines were treated with haloperidol in concentrations of 5, 10, 20, 50 and 100 μM for the U87 and U251 cell lines, and in concentrations of 10, 20, 40, 80 and 160 μΜ for the T98 cell line. Cell viability was evaluated by the trypan blue exclusion assay and 3--2,5-diphenyltetrazolium bromide  assay ,14,15. A4) were treated with haloperidol at various concentrations. As a negative control, cells treated with equal volumes of plain culture medium were used. All cell samples were run in triplicate in at least three independent experiments. For the DNA cell cycle analysis, the cells were harvested after incubation with trypsin, centrifuged, washed with phosphate-buffered saline solution (PBS), and incubated with propidium iodide (PI) working solution  at 37 °C in the dark for 20 min. Data from the PI fluorescence was collected to a total count of 10,000 nuclei, using a flow cytometer . Using the CellQuest software program (BD Biosciences) the cell cycle fractions G0/G1, S, G2/M, and sub-G0/G1 were analyzed as described previously [Cells . The migration distance at 0, 48 and 72 h after scratching was estimated for each cell line.Cultures of malignant glioma cells were treated with either haloperidol, TMZ, or a combination of haloperidol and TMZ, with or without radiotherapy. U87 and T98 cells were cultured in 24-well plates and after 24 h were treated with haloperidol and/or TMZ, then after a further 24 h irradiated at 4 Gy as previously described . Viabilip values < 0.05.The data were expressed as the mean ± standard deviation (SD). Two-way ANOVA with post hoc Tukey test for multiple comparisons was used to investigate the significance of differences between the results of different experimental conditions. Differences were considered significant at To determine the anti-glioma activity of haloperidol in GBM cells, U87, U251 and T98 cells were incubated with increasing haloperidol concentrations for 72 h. All the cell lines were sensitive to treatment with haloperidol in a dose-dependent manner. Using the trypan blue exclusion assay and MTT, the IC50 value of reduced viability for haloperidol was 23 μM in U87 cells, 35 μM in T98 and 38 μM in U251 cells a,b. On mTo investigate the cell cycle events underlying the observed growth inhibitory effects, we evaluated the effects of haloperidol on cell cycle progression in the U87 cell line. Cell cultures were treated with IC50 and twice the IC50 values of haloperidol for 72 h. Haloperidol induced a G2/M cell cycle arrest and an increase in the percentage of cells in sub G0/G1 in a dose dependent manner, suggesting the induction of apoptosis , Table 1To clarify whether the death observed under microscopy and in cell cycle analysis (increase in subG0/G1) was due to necrosis or to programmed cell death , the ability of haloperidol to promote an apoptotic effect in the U87 cell line was assessed. First, we analyzed whether haloperidol induced apoptotic phenomena during treatment for 12 and 24 h at IC50 values . FollowiTo investigate the expression of CD markers associated with migration, invasion and metastasis in U251 and T98 cells, 10,000 cells were seeded and after 24 h exposed to 100 μΜ haloperidol. Significant decrease in the expression of CD44 was observed in both cell lines. Decrease in the expression of CD24 was observed in both cell lines, but the decrease was statistically significant only in T98 .To investigate whether haloperidol could affect the migration of U87 and T98 glioma cells into a wound generated by scratching, a cell monolayer showed that, at concentrations of 20 and 40 μM for U87 cells, and at 40 and 80 μM for T98 cells, haloperidol significantly inhibited the wound recovery of both cell lines .To determine the effect of ionizing radiation in combination with haloperidol and/or TMZ, an escalating concentration of both drugs was administered to U87 and T98 cell lines. The IC50 values of TMZ were 50 μΜ in U87, and 160 μΜ in T98 cell lines. Cells were seeded and after 24 h were treated with haloperidol and/or TMZ, then 4 Gy radiotherapy was administered after a further 24 h. The MTT assay was conducted 24 h later. The effect of TMZ, as expected, was greater in U87 cell line. In both cell lines, the combination of haloperidol with TMZ and radiation therapy increased cell death .The present study demonstrated that haloperidol was an effective treatment for malignant glioma cells in vitro. Haloperidol induced the suppression of GBM cell growth, inhibited migration and produced cell cycle arrest in the G2/M phase. The induction of apoptosis was observed, with a significant decrease in the expression of CD44 and CD24 in the cells treated with haloperidol. The combination treatment of haloperidol, TMZ and radiation increased cell death. Cancer is the leading cause of death after cardiovascular disease. Although radiotherapy and chemotherapy have increased survival, the recurrence and side effects of treatment continue to limit their effectiveness, and new drugs that may improve current therapeutic options are urgently needed. To date, several studies have provided evidence that certain typical and atypical antipsychotic drugs may exert a chemotherapeutic effect in various forms of cancer ,19. EpidHaloperidol, a butyrophenone, is among the oldest antipsychotic drugs that are still used today. The main indications for its use include schizophrenia, delirium and bipolar disorder. This drug exerts its effect via strong antagonism, mainly of the D2 dopamine receptor. After the per os or intramuscular administration of haloperidol, the maximum concentration in plasma reaches 1–2 ng/mL and it is highly permeable in the central nervous system (CNS) . The conGBM is the most common malignant primary brain tumor in adults. Current treatment involves gross total surgical excision, followed by chemoradiotherapy. In spite of aggressive treatment, recurrence is an inevitable event and mean survival is in the range of 12–15 months . In GBM,We also investigated the expression of several CD markers, because they were implicated in cancer migration, invasion, and metastases, and they had not previously been studied in cancer cells treated with haloperidol. Haloperidol was associated with a significant decrease in CD24 and CD44, both of which have been related to cancer stem cells. CD24 positive tumors are reported to have a poor prognosis, and CD44 depletion has been shown to inhibit tumor proliferation and augment the effect of cytotoxic drugs ,37. ValpThe present study has several limitations. Plasma levels of haloperidol in patients with schizophrenia range from a low of 2 to 13 ng/mL to a high of 24.1 to 35 ng/mL . The dosOverall, given that GBM is a tumor that is difficult to treat, with the additional obstacle for many agents of the BBB, haloperidol appears to offer potential as an innovative treatment option. In view of the difficulty of finding novel anticancer drugs, drug repurposing constitutes a promising approach, given the known safety profile and the consequent diminished cost of phase I–II studies . There i


N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment.Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance.Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing.CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.The  Acute lymphoblastic leukemia (ALL) is a cancer of the blood and bone marrow. It is the most common childhood cancer, and its incidence is expected to increase worldwide , 2. OverVinca alkaloid vincristine (VCR) is a standard component in every combination chemotherapy regimen used to treat ALL , where I is the macroscopic current, V is the command potential, Vrev is the reversal potential (mV), Gmax is the maximal conductance (S), V0.5 is the half-activation potential (mV), and Vslope is the slope factor (mV/e-fold).To examine the voltage-dependent parameters of the sodium current, peak current amplitude from 25 ms test pulse potentials  were measured. Peak IV curves were fitted using: I50 values and neurite outgrowth assays were calculated using a two-way ANOVA followed by a Tukey’s post hoc test. A two-tailed, paired t-test was applied to compare intracellular levels of R123 between resistant and parental cells, as well as CSA-treated and non-treated cells. A two-tailed, paired t-test was also applied to compare current density between retinoic acid differentiated cells to all other experimental groups. All calculations were performed using GraphPad Prism v7.0 . Statistically significant results were represented by p < 0.05 .Statistical analysis of ICN-alkylisatins with varied substituents at the isatin nitrogen retain potency in drug resistant human lymphoma (U937) and uterine sarcoma (MES-SA) MDR cancer cell lines in vitro [N-alkylisatins  and 2ME2-resistant (CEM/2ME2-28.8R) ALL cell lines, and compared to the chemosensitive parental ALL cell line (CEM) using the MTS cell proliferation assay.We have previously demonstrated that 5,7-dibromo-in vitro . In ordeins Fig.  to retai6= 4.869, p < 0.005) decrease in sensitivity to VCR and were significantly cross-resistant to VBL, 2ME2, colchicine and paclitaxel (Table 8 = 4.529 p < 0.05) decrease in drug sensitivity. Interestingly, these cells did not demonstrate cross-resistance to VCR, VBL, colchicine or paclitaxel. Notably, both CEM-VCR R and CEM/2ME2-28.8R resistant cell lines treated with HMB-isatin, I4M-isatin or N2M-isatin showed a negligible change in IC50 values  after 48 h compared to their respective parental cells    differentiated SH-SY5Y cells and measured alterations in neurite length and number of branch points  , indicating a potential for lowered neurotoxicity.To further interrogate neurotoxicity, numbers of neurite branch points were examined following drug treatments Fig. b. At 1 nNeuronal morphology with neuritic-like processes in RA differentiated SH-SY5Y cells is associated with functional features that include transmembrane currents underlying a neuronal excitability phenotype –43. SuchNaïve SH-SY5Y (Ctr) cells displayed relatively small inward currents when compared to RA differentiated cells Fig. a. Treatm0.5: −18.6 ± 0.8 mV), only small depolarizing shifts on the activation midpoint (V0.5) were observed in VBL (V0.5: −13.3 ± 3.2 mV) treated, and naïve (V0.5: −13.8 ± 1.4 mV) cells. The latter also displaying an apparent increase in the slope of the activation curve . When compared to the RA group  and current density  in comparison to vehicle treated control cells , however these changes did not reach statistical significance  and the current density (measured from the ratio of peak current amplitude observed at 0 mV to the cell membrane capacitance in pA/pF) from each group. RA-differentiated SH-SY5Y exposed to HMB-isatin displayed modestly reduced conductance  highlights the need to further develop strategies to treat or prevent ALL relapse. The 5,7-dibromo-N-alkylisatins are a diverse and growing class of antimitotic agents that have demonstrated the ability to retain potency against clinically resistant cancer cell lines in vitro and reduce primary tumour growth in vivo [N-alkylisatins, namely HMB-isatin, I4M-isatin and N2M-isatin, to retain potency against drug resistant ALL cell lines. These N-alkylisatins also demonstrate a potential for reduced neurotoxicity compared to current clinically used Vinca alkaloids, VCR and VBL, suggesting that with further development, the N-alkylisatins could be used in the treatment of ALL with reduced potential for the onset of CIPN.ALL is the most common childhood malignancy and although high remission rates have been achieved, relapse occurs in ~ 20% of patients. This is predominantly due to the development of MDR to chemotherapy . Despite in vivo –30. The N-alkylisatin binding to β-tubulin, as any change in the tertiary binding site and/or its immediate environment may subsequently affect drug activity. In this study, cytotoxicity assays revealed that all three N-alkylisatins retained potency against the CEM/2ME2-28.8R cell line with IC50 values not significantly different to that of the CEM parental control cell line. Our competition binding experiments clearly indicate that N-alkylisatins compete for binding with a colchicine analog, strongly supporting their binding to the colchicine site. Therefore, the fact that the compounds retain potency against the 2ME2  resistaw et al.  and indiVinca alkaloids [50 values were not statistically different to those observed for the parental control CEM cell line. This was in contrast to the dose response profiles observed for the commercial anti-cancer agents and further confirms our findings [N-alkylisatins overcome MDR is yet to be uncovered, but we posit that polypharmacology could play a role [Multiple factors are thought to contribute to the drug resistant phenotype of the CEM-VCR R line , 48. Poilkaloids , howeverlkaloids , 51. LRPlkaloids . Clinicalkaloids . Moreovelkaloids , as welllkaloids . When thfindings  that thiy a role .N-alkylisatins to induce neurotoxicity by comparing their effect on SH-SY5Y neurite length and branch points to VCR and VBL, cytotoxins which are known to cause significant neuronal damage [Vinca alkaloids and N-alkylisatins displayed a dose-dependent effect towards the SH-SY5Y cells, however neurotoxicity associated with VCR and VBL treatment was significantly more pronounced. N-alkylisatins induced less damage to neurites than VCR and VBL, even at 10 × higher concentrations than the Vinca alkaloids. It is important to note that while the potency of VCR and VBL is between 2.7–13-fold greater than the N-alkylisatins against the chemosensitive CEM cell line, the N-alkylisatins were up to 17 × more potent than VCR and 2.3 × more potent than VBL in the CEM-VCR R resistant line, making the concentrations used in the neurotoxicity experiments highly relevant. Of the three N-alkylisatins tested, HMB-isatin was the least neurotoxic, and was therefore chosen for further assessment in functional patch-clamp assays.Antimitotic agents work by causing dynamic instability of microtubules and ultimately induce tumour cell death. They also interfere with the function of microtubules in axons which mediate neuronal vesicle transport –59. For l damage . DiffereFor neurofunctional experiments, we used differentiated SH-SY5Y cells as our model of neurons as they exhibit voltage-gated sodium channels essential to action potential generation and are therefore crucial for neuronal activity and cell survival. The cytotoxic drugs studied here affected sodium current magnitude with varied potency. Macroscopic conductance and current density are influenced by channel expression at the plasma membrane. Our patch-clamp results revealed that the average conductance and current density of HMB-isatin treated cells were not significantly different to those of RA differentiated control cells, which suggests that aspects of channel trafficking and expression in these two groups are akin. In contrast, the sodium currents of VBL and VCR treated cells were significantly smaller and similar in magnitude to the naïve group.v currents, RA and HMB-isatin, fire action potentials upon current injection. VBL treated cells, with somehow lower Nav current, also fired APs but only upon stronger stimuli (i.e. higher current injection), whereas VCR and naïve cells failed to fire under our experimental conditions. Spontaneous firings or trains of spikes in response to prolonged stimulation were not observed in any condition precluding rigorous quantification of the resulting excitability profiles. Perhaps the absence of (repetitive) firing is indicative of incomplete neuronal maturation and/or compromised cell fitness to support firing. Thus, our functional assessment points towards an inherent detrimental role of these drugs in differentiation and/or function of neuron-like SH-SY5Y cells.In our electrophysiological experiments, we observed complete inhibition of SH-SY5Y VGSC currents by 300 nM TTX confirming functional expression of only TTX-sensitive sodium channel isoforms . A detaiv channel behaviour and play critical roles in controlling neuronal excitability [Altogether, morphological and functional indicators from this study suggest that cytotoxic drug treatment affects differentiation towards the neuronal phenotype, as tubulin disrupting drugs, HMB-isatin, VBL and VCR possibly alter channel trafficking and/or expression. Other cytotoxic drugs like platinum compounds, contribute to the development of CIPN by impairing the electrophysiological function of dorsal root ganglion neurons demonstrated by a reduction and/or loss of the sensory action potential in nerve conduction studies . The redtability . Defectitability . Furthertability , 69. Our+ channels. The appearance of large inward TTX-sensitive Nav currents is an electrophysiological marker of neuronal-functionality. As a consequence, RA cells became excitable and thus fired action potentials in response to current injection (Fig. N-alkylisatins within a whole living organism [RA treatment significantly increased the density of the voltage-activated Naion Fig. a, insetsorganism .N-alkylisatin-based compounds with increased anti-cancer activity is ongoing and advancements in the field could lead to novel molecules that possess superior efficacy towards MDR cancers with lessened neurotoxicity profiles. A current opinion on how to best treat ALL relapse is to prevent it, which makes incorporation of these novel drugs into frontline therapy the most practical approach. Combining newer agents with conventional treatment approaches will likely overcome the challenge of balancing efficacy with toxicity and prevent the development of resistant ALL subclones. Altogether, the results obtained from this study reinforce the potential of N-alkylisatins to be considered as candidate drugs for the treatment of patients with relapsed and refractory ALL.The development of Additional file 1: Figure S1. Growth properties of CEM, CEM-VCR R and CEM/2ME2-28.8R cell lines. Figure S2. SH-SY5Y cell viability after treatment with HMB-isatin, VCR and VBL. Figure S3. Dose dependent effect of HMB-isatin and VCR on RA-differentiated SH-SY5Y neurite morphology. Table S1. Electrophysiological parameters of endogenous sodium currents. Table S2. Current density estimates from 3 independent experiments.


Compound 11c showed a broad spectrum in terms of antiproliferative efficacy with GI50 values in the range of 0.296 to 250 μM. Molecular docking studies indicated that Compounds 8a and 11c are accommodated in the colchicine binding site of tubulin in two different ways.Three series of fused pyrrolophenanthroline derivatives were designed as analogues of phenstatin and synthesized in two steps starting with 1,7-phenanthroline, 4,7-phenanthroline and 1,10-phenanthroline, respectively. Two (Compounds  Phenanthrolines are versatile moieties which have found use in many applications ,2,3 due At the same time, the fusion of two or more heterocycle rings can lead to novel classes of ligands, many with new interesting properties, including anticancer activity. For example, substituted benzo[c]phenanthroline analogues of nitidine and fagaronine showed cytotoxic properties associated with DNA intercalation and induced G2/M phases arrests . SynthetDespite the great progress achieved in chemotherapy, cancer remains a leading cause of mortality worldwide ,19. AmonEncouraged by the above considerations  and as part of our ongoing research aimed at investigating new anticancer drugs ,16,28, wWe furthermore considered some modifications at the 3,4,5-trimethoxyphenyl ring of phenstatin (ring A), generating either a 3,5-dimethoxyphenyl, 3,4-dimethoxyphenyl or a 4-bromophenyl ring.In this way, we incorporated the trimethoxyphenyl ring of phenstatin and our fused pyrrolophenanthroline system into a single molecule, in order to investigate their impact on the anticancer activity of the parent compound. Thus, we report here the synthesis and biological evaluation of novel compounds with 1,7-, 4,7-, and 1,10-phenanthroline scaffolds.5a–d were successfully synthesized using a two-step procedure starting from 1,7-phenanthroline 1. The first step comprised the formation of monoquaternary 1,7-phenanthrolin-7-ium salts 3a–d by the nucleophilic substitution of phenanthroline 1 to 2-bromoacetophenones 2a–d .4a–d from the corresponding salts 3a–d under triethylamine treatment. The in situ-formed ylides acted as 1,3-dipoles when reacted with ethyl propiolate, following a Huisgen 3 + 2 cycloaddition. Initially formed unstable intermediates 5′a–d undergo an aromatization process under the current reaction conditions, leading to target compounds 5a–d in 50–88% yields  with ethyl propiolate  were identified by NMR and IR. Compounds ted data ,31,32,335a–d, 8a–d and 11a–d were submitted to the National Cancer Institute  and four compounds were selected for the evaluation of their antiproliferative activity against their panel of 60 human cancer cell lines. The NCI panel is organized into nine sub-panels representing leukemia, lung, colon, central nervous system (CNS), melanoma, ovary, kidney, breast and prostate cancer cells. The panel also includes several multidrug-resistant tumor cell lines . The four compounds—5c, 8a, 8b and 11c—selected by NCI were first tested at a single high dose (10−5 M) in the full 60-cell panel, selected results being presented in 8a and 11c showed very good growth inhibition of several cancer cell lines. The best efficacy in terms of growth inhibition was shown by Compound 11c against the HL-60(TB) RPMI-8226 leukemia cell line, NCI-H522 non-small cell lung cancer line, COLO205 and HT-29 colon cancer cell line, SF-539 CNS cancer cell line, MDA-MB-435 and M14 melanoma cell lines, OVCAR-3 ovarian cancer cell line and A498 renal cancer cell line, these results being comparable or better than phenstatin, which was used as a control. Compound 11c also showed cytotoxic activity against the COLO205, MDA-MB-435 and A498 cell lines. An important growth inhibition effect was also observed for Compound 8a against MDA-MB-435 melanoma cells and K-562, SR and HL-60(TB) leukemia cells. Thus, replacement of ring B of phenstatin with a pyrrolo[c]phenanthroline or a pyrrolophenanthroline group retains the growth inhibition properties of phenstatin. The more active Compound 8a, by comparison with 8b, retains the three methoxy groups of the more active phenstatin. Therefore, the lack in the methoxy substituent in para position of the ring B appears to be not favorable for the anticancer activity, at least in the case of compounds type 8.Compounds lophenanthr11c, which had the best growth inhibition profile among the tested compounds, progressed to the full five-dose assay. Selected GI50 values are presented in 11c possess excellent to moderate antiproliferative activity with GI50 values ranging from 0.296 to 3.78 μM against 40 cancer cell lines from all nine sub-panels. In particular, Compound 11c showed the best GI50 and TGI  values (296 nM and 981 nM respectively) against MDA-MB-435 melanoma cells. Promising GI50 values were also obtained for against NCI-H522 lung cancer cells, HCT-116 colon cancer cells and M14 melanoma cells. However, when comparing to control phenstatin, there are only a few cell lines against which Compound 11c is more potent  or shows similar antiproliferative activity .Compound 8a and 11c and the lack of activity in the case of 5c and 8b.In order to verify if our target phenstatin analogues retain the ability of their parent compound to fit in the colchicine binding site of tubulin, docking studies were performed on the four NCI-tested compounds in the colchicine binding site of the α,β-tubulin heterodimer (PDB:1SA0). The in silico study aimed to evaluate the shape and electrostatic complementarity between the tested ligands and the α,β-tubulin heterodimer interface, which could account for the observed antiproliferative effects in the case of 8a and 8b in the colchicine binding site of tubulin revealed a similar accommodation to previously reported ligands phenanthroline group appears to be the most beneficial, while the replacement with a pyrrolo[8 and 11 are amenable for further structural optimization in the development of chemotherapeutic agents.Three new classes of potential phenstatin analogues have been synthesized and characterized. Four compounds were selected by NCI and tested against a panel of 60 cell lines at a single concentration of 10


Ferula L., belonging to the Apiaceae family, dicotyledonous plants present in many temperate zones of our planet. Ferula communis L. is the main source of sesquiterpene ferutinin, a bioactive compound studied both in vitro and in vivo, because of different effects, such as phytoestrogenic, antioxidant, anti-inflammatory, but also antiproliferative and cytotoxic activity, performed in a dose-dependent and cell-dependent way. The present review will focus on the molecular mechanisms involved in the different activities of Ferutinin, starting from its antioxidant potential at low doses until its ionophoric property and the subsequent mitochondrial dysfunction induced through administration of high doses, which represent the key point of its anticancer action. Furthermore, we will summarize the data acquired from some experimental studies on different cell types and on several diseases. The results obtained showed an important antioxidant and phytoestrogenic regulation with lack of typical side effects related to estrogenic therapy. The preferential cell death induction for tumor cell lines suggests that ferutinin may have anti-neoplastic properties, and may be used as an antiproliferative and cytotoxic agent in an estrogen dependent and independent manner. Nevertheless, more data are needed to clearly understand the effect of ferutinin in animals before using it as a phytoestrogen or anticancer drug.The employment studies of natural extracts in the prevention and treatment of several diseases highlighted the role of different species of genus  The use of herbal and plants extracts to prevent and to treat several acute and chronic diseases is an object of growing interest and investigation in pharmacological and nutraceutical research ,2,3.In vitro and in vivo evidence showed that the bioactive compounds isolated from herbs and plants showed antioxidant, anti-inflammatory and anti-cancer property ,5,6. The oxidative-stress reduction leads to a decreased risk of onset of several pathologies, such as cancer and chronic inflammatory diseases . Among tThe results obtained in different experimental works suggest that the sesquiterpenes have protective potential at low doses and cause severe toxic effects at high doses ; this biFerula L. is one of the most representative .,83.82,83Increasing evidence on the ability of ferutinin to inhibit cell growth and to induce apoptosis both in vitro and in vivo  has led In addition, the SMAC/DIABLO molecule, inhibitor of the inhibitors of apoptosis proteins (IAPs), might be involved in the mechanism of ferutinin toxicity, as once leaked from the mitochondria, it can bind IAP, preventing the inhibition of caspase activity and promoting the formation and activation of the apoptosome complex, leading to caspase-9 activation ,91 that Among cell lines tested to verify ferutinin effects, it was observed that, for tumor cell lines, human breast adenocarcinoma cell line (MCF-7), human cells of urothelial carcinoma (TCC), human colon adenocarcinoma cells (HT29), murine cells of colon carcinoma (CT26), IC 50 was between 67 and 81 μM, while the IC 50 for normal human and murine fibroblast lines (HFF3 and NIH/3T3) was higher, with values of 98 and 136 μM respectively, with about 67% of viable cells in both cell lines treated at the above concentrations  32,33,3,35,36. AFurthermore, Arghiani et al. (2015) observed cytotoxic effects of ferutinin on human teratocarcinoma (NTERA2) and oesophageal cancer (KYSE30) cell lines and the IC50 values were respectively 39 μM and 58 μM .The anti-cancer ferutinin activity was also evaluated in vivo, in comparison with cisplatin, on tumors developed in hybrid BALB/C mice: a tumor reduction of 67% for ferutinin and of 72% for cisplatin was observed; however while the latter produced a significant alteration of the liver tissues and the spleen of the treated mice, this did not happen for mice in treatment with ferutinin . In vitro studies have shown that, thanks to its phytoestrogenic activity, ferutinin induced the inhibition of proliferation in human breast carcinoma MCF-7 cells . To testA recent study confirmed the cytotoxic activity of ferutinin also on the prostate cancer (PC-3) cell-line, with IC 50 values of 16.7 µM. The low IC 50 is due to the cross-link among the cytotoxic activity and hormonal action, since the prostate cancer onset is primarily due to androgenic hormones .Gao et al. (2013) showed that ferutinin, in a dose-dependent way and, particularly, at high concentrations (40 μM or above), was able to induce apoptosis in human red blood cells (RBCs), without the involvement of mitochondria since these cells lose those organells during the maturation process. The erythroptosis is mediated by simultaneous increase of the cytosolic free calcium ions level and the activity of caspase-3. In addition, the external calcium depletion had not blocked the apoptosis mediated by ferutinin, suggesting that the existence of unknown mediators of apoptosis are ferutinin-induced, which could be added to the ionophoric activity . CertainFerula genus extract contains a high percentage of the bioactive compound ferutinin, having a wide range of biological effects that elect it as a possible therapeutic agent with a broad spectrum of action. We examined the effects of ferutinin, highlighting the deep gap existing between the uses of high and low doses.The Indeed, the ferutinin administration at low-concentration is associated with an important antioxidant action, although the mechanisms of free radical decrease still need further clarification.Moreover, the effects of cross-talk between the estrogenic and ionophoric properties of ferutinin that lead to strongly reduction of the side effects produced by usual estrogen therapy, allow this molecule to be eligible as a potential therapeutic agent in the gynecological pathologies. Concerning the antiproliferative action of ferutinin, several in vitro and in vivo studies showed that the bioactive compound acts both on cytotoxicity and cell proliferation in an estrogen-dependent and -independent manner and its activity is dose dependent and variable according to the cell type analysed, with a preferential action for tumor cell lines.The ionophoric effect of ferutinin with the consequent loss of the trans-membrane potential seems to be the key point of the antiproliferative and cytotoxic effect, both directly through the activation of mitochondrial-dependent apoptosis and indirectly through the effects produced by the summarization of NO and ROS, molecules that play a key role in the production of stress, senescence and death signals.Therefore, based on the evidences obtained, the bioactive compound ferutinin can be considered an effective therapeutic agent as adjuvant or substitute for the current anti-neoplastic treatments. More studies should be performed to evaluate its action on other cell types and further molecular pathways involved, to compare its activity with that of chemotherapy drugs going to evaluate the different degree of toxicity produced as a side effect on healthy cells.


A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer’s disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A . High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation  upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD. Acute inflammation plays an essential role in the normal response to tissue injury3. This inflammatory response initiates a cascade of cellular activation signals in innate immune cells , resulting in increased production of proinflammatory cytokines and chemokines6. These cytokines and chemokines are essential to the recruitment and activation of other cells in the innate and adaptive immune systems. An inappropriate response to inflammation or alterations in the production of these chemokines can result in disease development.Inflammation is associated with increased risk of developing cancer, autoimmune disorders, and neurodegenerative diseases such as Alzheimer’s disease (AD)7. Significant progress has been made towards understanding the genetic architecture of AD. To date, 32 loci have been identified with common and rare alleles that influence the risk of AD10. AD is pathologically characterized by the accumulation of amyloid beta (Aβ) peptide forming insoluble aggregated plaques in the gray matter and is thought to result in neurodegeneration and loss of memory function11. However, the characterization of biological mechanisms by which these rare variants and the accumulation of Aβ modulate risk for disease has proven to be extremely difficult, with only several of these variants being experimentally characterized19.AD is the most common neurodegenerative disorder and is the main cause of dementia in the elderly population20. This genome-wide association study (GWAS) of 59 AD-related CSF analytes used two independent datasets: the Knight Alzheimer’s Disease Research Center (ADRC) and Alzheimer’s Disease Neuroimaging Initiative (ADNI)20. This study demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with atypical chemokine receptor chemokine-binding protein 2 (ACKR2) variant V41A.Recent analyses of quantitative AD endophenotypes in cerebrospinal fluid (CSF) has identified a chemokine receptor variant that alters the risk and rate of progression of ADCCL2 gene located on chromosome 17q11.2-q12. CCL2 is a pro-inflammatory chemokine involved in recruitment of immune cells from the blood to sites of inflammation via chemokine gradients. CCL2 helps to control blood brain barrier migration of monocytes and dendritic cells and aids in the differentiation and migration of macrophages21. In the brain, CCL2 is mainly secreted by astrocytes, microglia, and in low levels by endothelial cells22. Upon an immune response to infection, injury or inflammation, CCL2 is produced in the central nervous system (CNS)23. CCL2 interacts with chemokine receptor CCR2 which signals via a G couple protein cascade. Upon CCR2 and CCL2 ligand interaction, a cascade of cell activation events takes place . This activation cascade signals cell migration, cell survival, transcription regulation, and release of pro-nociceptive molecules25. Upon CCL2 binding to a receptor, it induces a strong chemotactic response and mobilization of intracellular calcium ions and synaptic network activity in the hippocampal neurons27. High levels of CCL2 is a risk factor in several neuroinflammatory and neurodegenerative brain diseases such as multiple sclerosis, brain ischemia, traumatic brain damage, and AD28. In AD mouse models, CCL2 is key to inducing chronic inflammation and activation of immune cells, and secretion of other chemokines29. Overexpression of CCL2 retains activated microglia cells near the inflamed site30. This results in increased interactions of activated microglia with key features of AD: amyloid plaques, plague aggregation, and cognitive decline33. Inhibition or removal of CCL2 in AD mouse models revealed accelerated amyloid pathology formation34. These studies demonstrate that proper function and regulation of CCL2 is imperative to preserve brain innate immune response homeostasis and cognitive function.CCL2 is also known as monocyte chemotactic protein-1 (MCP-1) and is encoded by the 35) is encoded by the ACKR2 gene located on chromosome 3p21.3. ACKR2 is a seven transmembrane G-protein-coupled receptor containing three intracellular loops and three extracellular loops36. ACKR2 binds with the 14 inflammatory chemokines of the CC family, including CCL2 and CCL4, but not CCL1937. ACKR2 is an atypical chemokine due to its lack of a canonical DRYLAIV motif in the second extracellular loop. This lack of motif inhibits the ability of the receptor to signal the production of other cytokines36. Instead, ACKR2 recognizes inflammatory chemokines, scavenges them, and upregulates the production of more ACKR2 receptor38. ACKR2 plays an essential role in the regulation of the inflammatory response by internalizing inflammatory chemokines, facilitating their destruction when the cellular endosome fuses with an acidic lysosome. The chemokine-free ACKR2 receptor is then recycled back to the cell surface and can scavenge additional inflammatory chemokines39. ACKR2 is mainly expressed on leukocytes, including dendritic cells, monocytes, macrophages, and innate-like B cells40. In the central nervous system (CNS) ACKR2 is mainly expressed in microglia and astrocyte cells43. Nonsynonymous mutations mapped to the ACKR2 gene have been associated with increased risk for breast cancer, testicular leukemia, CD45 deficiency, glucose intolerance and inhibit leukocyte type differentiations and monocyte counts49.Atypical chemokine receptor 2 , rs2228467 was associated with increased coronary heart disease and a higher monocyte count in the blood stream51. Rs2228467 was also identified as an increased risk factor for developmental disorders following the analysis of over 700 mother-infant pair mid-gestational serum and neonatal bloodspots52. Another 2017 study using the National Human Genome Research Institution (NHGRI) dataset found that rs2228467 is associated with increased AD risk53. Together these studies make a compelling case for the biological characterization of ACKR2-V41A. Here we provide the molecular and biological characterization of ACKR2-V41A and show that it has decreased binding affinity, scavenging efficiency, and receptor upregulation  for CCL2. These results provide insights into the previously reported association of ACKR2-V41A with AD risk, rate of cognitive decline, and serum markers of inflammation which could lead to novel therapeutic solutions for AD.The ACKR2-V41A allele (rs2228467) is associated with altered chemokine levels in the CSF and bloodstream. A 2017 study with 8,293 Finn participants identified increased levels of the chemokine eotaxin (a potent eosinophil attractant) associated with rs2228467 and increased risk of developing Crohn’s disease, multiple sclerosis, and ulcerative colitisACKR2-V41A differs from wild type by a single amino acid located at the chemokine binding site of ACKR2. V41A is specifically located in the first of three extracellular loops. Predicted protein folding models were generated and ACKR2-WT and ACKR2-V41A were superimposed to check for differences in folding and hydrophilicity. There was no observed difference in predicted protein structure folding Fig. .Figure 1RegulomeDB gave this nonsynonymous mutation a score of 5/6. PolyPhen-2 predicted this variant to be probably damaging with a score of 0.958 . These predictor tools - Chimera, RegulomeDB and PolyPhen-2 all predicted that the ACKR2-V41A variant causes a biological change with functional consequences.The single amino acid change at position 41 is predicted to have a slight decreased hydrophilicity between ACKR2-WT and ACKR2-V41A Fig. . DespiteCHO-k1 cells were transfected with ACKR2-WT or ACKR2-V41A and were grown in selective media for a week before transfection positive cells were enriched using beads and magnetic columns. The enriched CHO-k1 ACKR2-WT or ACKR2-V41A cell populations had similar percentages of receptor positive cells Fig.  and receThe binding affinity of ACKR2-WT and ACKR2-V41A for CCL2 was measured in both a direct binding and a competition assay. In the direct binding assay, the percentage of CCL2 binding to ACKR2-WT and ACKR2-V41A was determined by measuring the levels of AF647-conjugated CCL2 divided by the ACKR2 receptor levels. Analysis of 5 independent runs revealed that ACKR2-V41A has a significantly lower binding affinity than ACKR2-WT Fig. . Non-linA competition assay was also performed to test if the unconjugated chemokine ligand CCL2 was able to interfere with binding of an anti-ACKR2 antibody. Analysis of 5 independent runs showed a significant difference between chemokine receptor ACKR2-WT and ACKR2-V41A . ACKR2-V41A expressing cells bound to significantly lower levels of CCL2, allowing the ACKR2 antibody to bind at a higher percentage (μ=88.5) than ACKR2-WT (μ = 66.18). Quantification of levels of unconjugated CCL2 blocking the anti-ACKR2 antibody show that ACKR2-V41A (11.5%) binds with significantly lower affinity to CCL2 than ACKR2-WT (33.82%)  than ACKR2-WT (26.0%) Fig. . Under t50 of 18.19 hours, illustrating that the WT receptor scavenges CCL2 more efficiently than ACKR2-V41A, which has an IC50 of 36.83 hours  induced by CCL2. Upon activation of the receptors with chemokine ligand CCL2, cells expressing ACKR2-V41A receptor had significantly lower p-cofilin levels than cells expressing ACKR2-WT  imaging. Upon activating the receptor with chemokine CCL2, cells expressing the ACKR2-V41A receptor have significantly lower levels of calcium signaling starting 4 minutes after CCL2 activation when compared to CCBP-WT  and a possible mechanism for increased levels of CCL2 in the CSF. ACKR2-V41A has lower CCL2 binding affinity, CCL2 scavenging efficiency, and CCL2 induced ACKR2 receptor upregulation. As proposed in the original GWAS analysisChimera protein predictor software, PolyPhen-2, and Regulomedb predicted that the amino acid change from the hydrophobic valine to the less hydrophobic alanine was sufficient to change biological function. In this study, we demonstrated that variant V41A, located at the first extracellular loop and binding site, reduces the binding affinity of receptor ACKR2 with CCL2 and CCL4 ligands. We suspect that same behavior will be observed if the binding affinity of this variant is tested with the remaining 12 chemokine ligands known to interact with it. In addition, ACKR2-WT and ACKR2-V41A did not noticeably improve binding for the chemokine ligand CCL19. This ligand is one of the chemokines from the CC family that does not interact with ACKR2 receptor. Thus, in this case the V41A variant did not enable the ACKR2 receptor to interact with a new chemokine ligand.This altered ability of ACKR2 to bind CCL2 resulted in decreased CCL2 scavenging activity and lower levels of receptor on the cell surface. ACKR2-V41A lost its scavenging efficiency over time (after 18 hours of saturating the system with 100 pg/ml of CCL2) and also has decreased levels of the ACKR2 receptor at the cell surface over time (starting at 18 hours after the cells were stimulated with 100 pg/ml of CCL2) compared to wild type. We found that ACKR2-V41A is two-fold less efficient at scavenging CCL2, resulting in the maintenance of this inflammatory chemokine signal for a longer time than wild type. These results suggest that binding affinity and receptor levels are key to regulating levels of pro-inflammatory chemokines such as CCL2.55. Here we report that after CCL2 activation, p-cofilin expression levels were reduced in cells with the ACKR2-V41A receptor compared to wild type. This result aligns with the assay examining ACKR2 receptor levels after CCL2 stimulation over a 72 hours period. Low levels of p-cofilin in ACKR2-V41A cells could explain the low level of ACKR2 quantified at the cell surface and the decreased CCL2 scavenging activity. In addition, there are compelling studies showing that ACKR2 is capable of Ca2+ mobilization upon stimulation with CCL2 in in HEK 293 cells56 and neurons57. These studies align with our results and strengthen our proposed mechanism. Here we have shown a reduction of the biological capacity of the atypical chemokine ACKR2-V41A to bind, scavenge, and be activated by CCL2. Additional impacts of the ACKR2-V41A allele should also be examined in regard to the other 12 chemokines ACKR2 is known to bind with37. Thus, alterations in chemokine levels caused by ACKR2-V41A have a powerful effect on chemokine gradient homeostasis, inflammation, and increased risk to develop AD.ACKR2 is an atypical chemokine receptor and lacks the signaling motif to initiate the production of other chemokine and cell migration. ACKR2 stimulation by CCL2 activates the β-Arrestin-dependent signaling pathway cofilin pathway [Rac1-p21-activated kinase 1 (PACK1)-LIM kinase 1 (LIMK1) cascade] and ACKR2 can send an intracellular signal to upregulate surface receptor levels59. Thus, regulation of CCL2 levels has multiple effects on brain homeostasis and longevity. In this study, we observed significant differences in the ability of ACKR2-V41A to bind and scavenge CCL2, potentially resulting in higher levels of CCL2 at the injury site and maintenance of pro-inflammatory signaling and monocyte recruitment, which may lead to increased Aβ plaque aggregation and risk for AD.In the presence of Aβ aggregation the expression and production of pro-inflammatory related molecules, including CCL2, are elevated. In this context, it is known that high levels of CCL2 expression and production trigger microgliosis, increase the APP/CCL2 ratio, and increase Aβ plaque aggregation. CCL2 could increase the rate of Aβ deposition by interference with clearance as well as increase expression of apolipoprotein E, a main biomarker for AD pathogenesis and Aβ deposition60. Consequently, genetic variants identified by GWAS have to be physiologically significant to conclusively identify their biological effects as proteins. In this study we provide the biological characterization of ACKR2-V41A that was identified using a GWAS. We have provided insights into the mechanism by which ACKR2-V41A with CCL2 alters the chemokine gradient homeostasis, increases inflammation, and the alters risk for AD. Future efforts should focus understanding if and how ACKR2-V41A impacts Aβ production and aggregation and other biomarkers of AD in AD-relevant cell lines.GWAS researchers have identified thousands of genetic variants contributing to the rate of development of complex human diseases; providing insights at the DNA level of how a single nucleotide polymorphism (SNP) might cause changes in function at the biological and molecular level. The characterization of biological mechanisms by which rare variants found in GWAS to modulate risk for disease has proven to be extremely difficult. SNP characterization requires a deeper understanding of how DNA changes might affect gene expression through transcription, RNA splicing, ligand-receptor binding, protein function etc62. These 11 models were compared to similar chemokine receptors for accuracy. Using UCSF Chimera Software, the 3D models were oriented in space63. We superimposed ACKR2-WT and ACKR2-V41A to check for differences in folding and hydrophilicity. 3D images were produced using UCSF Chimera Software to highlight the predicted chemokine receptor structure and hydrophobic interactions at amino acid position 4163.ACKR2-WT and ACKR2-V41A chemokine receptor amino acids sequences were sent in a FASTA format to the University of Michigan’s Zhang Lab LOMETS server. 3D models were generated by collecting high-scoring structural templates from 11 locally-installed threading programs 64. This predicter algorithm scores between 1 and 6 and any variant categorized between 1 and 5 is predicted to have damaging biological function in the human genome. PolyPhen-2 is an algorithm that predicts possible biological impact of an amino acid substitution in the human genome65. This algorithm classifies the biological impact of a variant from a damaging to a tolerable variant.Annotation and prediction of the biological function of ACKR2-V41A variant was determined using RegulomeDB and Polymorphims Phennotyping v2 (PolyPhen-2). RegulomeDB is a database that annotates variants with known and predicted regulatory elements in human genome such as eQTL, transcription factor binding and transcription factor motifa cells, amplified, and purified using the ZymoPURE II Plasmid Maxiprep kit (Cat #D4203). Plasmids were transfected into Chinese Hamster Ovary (CHO-k1 ATCC® CCL-61) cells using lipofectamine  and manufacture recommendations were followed. Properly transfected cells were selected using the antibiotic G418 sulfate . Transfected cells were grown in F12 media  with 10% FBS (HYCLone Cat #SH30071.01). Cell media was changed every 48 to 72 hours depending on cell confluency levels.Plasmid construct were ordered at Genscript. DNA sequences for ACKR2-WT and ACKR2-V41A were inserted into vector pCMV6-AN-myc-DDK . Transformation protocols were followed as recommended by the manufacturer. Briefly, plasmids were transformed into DH5We tested the atypical chemokine receptor ACKR2-V41A function using CHO-k1 cells because they lack expression of the wild type ACKR2 receptor as well as other competing human chemokine receptors. It is important to note that this cell type is absent in the human brain and therefore our results should not be overinterpreted. After transfecting CHO-k1 cells with plasmids carrying ACKR2-WT or ACKR2-V41A, cells were incubated until they reached 80% confluency and prepared for cell sorting. Cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies (ABNOVA Cat #H00001238-B02). After two washes, cells were stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE (Biolegend Cat #405307). Cells were then washed and isolated using anti-PE MicroBeads from MACS Miltenyi Biotech (Cat #130-048-801) following manufacture recommendations. After isolation, cells were incubated in F12 media until 80% confluency was reached.2 flask reached 80% cell confluency. 250,000 cells were resuspended in 300 μL of PBS-1% BSA and placed in FACs tubes. To evaluate expression of the ACKR2 receptor, cells were stained with anti-CD16/32 (Fc block) on ice for 20 minutes . After two washes, cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies, washed twice, and then stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE. After two washes receptor expression was analyzed on a BD Accuri. In addition, expression of the receptor at cell surface was monitor for 72 hours after CCL2 was added to the growing media. Expression levels were obtained following the procedures above.CHO-k1 cells transfected with ACKR2-WT or ACKR2-V41A were harvested when a 75 cmTo determine CCL2 binding levels for ACKR2-WT and ACKR2-V41A, cells were incubated with conjugated CCL2-AF647  at 37 °C for 45 minutes at different concentrations ranging between 100 to 750 nM. After two washes receptor expression was analyzed on a BD Accuri. To determine the levels of ACKR2-WT and ACKR2-V41A receptor expression, cells were stained with anti-CD16/32 (Fc block) on ice for 20 minutes. After two washes, cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies, washed twice, and then stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE. In the CCL2 competitive binding assay levels for ACKR2-WT and ACKR2-V41A, cells were incubated with unconjugated CCL2  at 37 °C for 45 minutes. After two washes and an Fc block as described above, cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies, washed twice, and then stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE. After two washes, levels of binding of the ACKR2 antibody was analyzed in BD Accuri.CCL4  competition binding assay was performed following the procedures above. Conjugated CCL19-AF647  binding analyses were completed using the same methods used for CCL2 binding assay.ACKR2-WT and ACKR2-V41A cells were seeded in a 6-well cell culture plate with fresh F12 media with 10% FBS and 100 pg/mL of CCL2 added to each well. At varying time points, 150 μL of media supernatant was extracted from each well to measure the remaining concentration of CCL2 using a CCL2 TMP sandwich ELISA kit . Ninty-six well ELISA microplates were coated overnight at room temperature with a monoclonal antibody specific for human CCL2 (0.25 μg/mL in PBS). Plates were washed three time with 0.05% tween diluted in PBS and then wells incubated with blocking buffer (PBS/1% BSA) for 2 hours at room temperature. After three washes, samples and controls were added to the wells and incubated for 2 hours at room temperature. After three washes plates were incubated for 1 hour at room temperature with biotinylated anti-human CCL2 antibody (0.25 μg/mL) diluted in blocking buffer. After three washes, plates were incubated with 100 μL of Streptavidin-HRP conjugate (0.05 μg/mL) for 30 minutes at room temperature. Plates were washed three times and 100 μL of TMB substrate solution (hydroperoxide and chromogen tetramethylbenzidine) was added for 20 minutes at room temperature in the dark. The reaction was stopped with 100 μL of 1 M of hydrochloric acid. Optical density was determined using a micro plate reader set at 450 nm with a wavelength correction of 620 nm.55. Briefly, 300,000 CHO-k1 cells expressing either ACKR2-WT or ACKR2-V41A were seeded in a well and serum-starved for 18 hours. Then, cells were stimulated with 100 nM of CCL2  and incubated at 37 °C in 5% CO2 for 30 minutes. Cells were harvest, lysed using RIPA lysing buffer  following manufacture recommendations. Lysed product was prepared for a western blot assay. Two gels were run in parallel using the same lysed product samples. One gel was stained with antibody for p-cofilin  to detect phosphorylated cofilin. ImageJ  was used to analyze the intensity of bands corresponding to p-cofilin. The second gel was stained with cofilin  and was used for normalization of p-cofilin after ImageJ analysis. An independent T test was performed to determine if p-cofilin levels differ between cell expressing ACKR2-WT and ACKR2-V41A.Levels of phosphorylated cofilin were obtained following methodology from previously described research2. The next day, cells were loaded with 4 μM of Fura-2AM  in Ringers solution  for thirty minutes at 37 °C in 5% CO2. Cells were then washed once with Ringers solution and incubated for another 30 minutes at 37 °C in Ringers solution. Calcium imaging was performed at room temperature using an Olympus IX51 inverted microscope equipped with a xenon arc lamp. Fura-2AM loaded CHO-k1 cells expressing receptor ACKR2-WT and ACKR2-V41A were excited using 340 nm and 380 nm excitation filters, and images of 340 nm, 380 nm, and transmitted light was captured using a fluorescence microscope camera (Q Imaging Exi Blue) with a 20x objective (N.A. 0.75) at 1.6-sec intervals. At the one minute time point in each imaging protocol, 250 nM of recombinant Human CCL2 was added to stimulate Ca2+ flux23. Ionomycin  was added at the five minute time point as a positive control. Ten to twenty representative cells were selected as regions of interest in each frame, and F340:F380 ratios were calculated and analyzed using CellSens software from Olympus. Each individual cell’s fluorescence was normalized to its first recorded value according to the equation (F-Fo)/Fo, where F is the fluorescence at the specific time point, and Fo is the fluorescence value at time 067.Cells were seeded on a 8-chambered slide  and incubated overnight in F12 media with 10% FBS at 37 °C in 5% CO50, and IC50 were calculated using non-linear analysis best fit test. Each independent run contained a minimum of three replicates per sample and the mean of those replicates were considered a single point of analysis. In this study, each experiment was performed with three to five independent runs (9 to 15 replicates per sample). Results presented are representative of observed phenotypes and analysis with P-values less than 0.05 were considered significant.Statistical analyses were performed using the Prism 8 software . Differences in binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity between ACKR2-V41A and ACKR2-WT were analyzed using an unpaired T-test. Kd, ECSupplementary information.