Panel Of Tumor Cell Lines Research Articles

Abstract LB536: Newly established CAR-T target antigen luciferase reporter cell lines facilitate CAR-T development

Abstract Adoptive T cell therapy is a type of immunotherapy in which T cells are genetically modified to fight tumors. It has emerged as an exciting new approach for cancer treatment. Chimeric antigen receptor (CAR)-T cells have displayed remarkable efficacy in treating malignant cancers, particularly liquid tumors. CAR-T cells have proven to be a new type of ’living’ therapeutic by harnessing the patients' immune system to recognize specific tumor associated antigens and redirect the engineered T cells to more specifically target tumor cells. A large number of research efforts have been put into developing new CAR structures to increase the scope of targeted cancers and anti-tumor efficacy. Evaluating the biofunction of CAR-T cells in vitro often involves a series of labor-intensive co-culture experiments and immunoassays. Reproducibility also remains a challenge during the validation of new CAR-T cells due to donor-to-donor variation and other factors. In this study, we present a panel of luciferase reporter tumor cell lines that can be utilized to examine the function of CAR-T cells. The panel of selected human tumor cell lines naturally express high levels of CAR-T target antigens on cell surface, such as CD19, CD20 and Her2. After introduction of a Lenti-LUC2 luciferase reporter into the parental cell lines, single cell cloning was performed to isolate stable clones with high luciferase expression. To verify the performance of the target Luciferase reporter cell lines, we used commercially available CAR-T cells expressing CD19, CD20, HER2 and empty vector-transduced T cells as controls in co-culture experiments. The cytotoxicity of the CAR-T cells against target tumor cells was measured using both a luciferase assay and the xCELLigence potency assay. Our results demonstrate that the luciferase reporter system is a relatively simple, robust, and highly sensitive means to measure biological processes. The luciferase reporter cell lines provide an advantage in measuring target cell killing without having to use radioactive 51Cr release assay or pre-labeling the cells in CAR-T functional evaluation. In addition, these reporter cell lines were characterized and authenticated using cell morphology, growth kinetics, and STR analysis. The expression stability of both the target antigen and luciferase was verified by comparing the low passage and the high passage reporter cells. In summary, the well-characterized CAR-T target antigen luciferase reporter cell lines from ATCC provide excellent tools for studying CAR-T biofunction and validating new CAR-T agents for cancer immunotherapy. Citation Format: John Foulke, Brian Della Fera, Luping Chen, Fang Tian. Newly established CAR-T target antigen luciferase reporter cell lines facilitate CAR-T development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB536.

Abstract 1881: Drug combination screening of Ipatasertib and Abemaciclib with other targeted agents in complex multicellular tumor spheroids from the NCI-60 and the National Cancer Institute’s Patient-Derived Models Repository

Abstract Drug combinations are frequently used to improve clinical efficacy, to minimize toxicity, and to reduce the development of drug resistance. Here, we investigated the growth inhibitory activities of the pan-Akt inhibitor ipatasertib and the CDK4/6 inhibitor abemaciclib in combination with other targeted agents. Twelve well-characterized patient-derived cancer cell lines from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm) and seven established cell lines from the NCI-60 tumor cell line panel (https://dtp.cancer.gov/discovery_development/nci-60/cell_list.htm) were grown as multicellular 3D complex spheroids. The complex spheroids, a mixture of tumor cells (60%), endothelial cells (25%), and mesenchymal stem cells (15%), were grown for 3 days before drug(s) were added. All agents were tested both alone and in combinations at multiple concentrations up to their reported clinical Cmax value and cell viability was assayed using CellTiter-Glo 3D seven days after drug exposure. While abemaciclib had minimal activity as a single agent, ipatasertib was noticeably selective for tumor cells harboring activating PI3K/AKT/mTOR pathway variants. Dual inhibition of the PI3K/AKT/mTOR and RAS/MEK/ERK pathways was one of the most effective combinations. For example, the combination of ipatasertib with the MEK inhibitor selumetinib or the ERK inhibitor ravoxertinib resulted in additive and/or synergistic cytotoxicity in over half the complex spheroid models screened. The V600E variant-specific BRAF inhibitor vemurafenib and the KRAS G12C selective inhibitor sotorasib in combination with ipatasertib showed activity in the one BRAF V600E and two KRAS G12C variant containing complex spheroid models, respectively. Another effective ipatasertib combination was vertical inhibition of the PI3K/AKT/mTOR pathway with the mTORC1/2 kinase inhibitor sapanisertib, which demonstrated additive and/or synergistic responses across multiple complex spheroid models. For abemaciclib, the most successful combination was with the CDK2/7/9 inhibitor BMS-387032, which achieved greater than one log of cytotoxicity in the majority of the complex spheroid models. For the combination of abemaciclib and selumetinib, there was a high correlation between the responses of two patient-derived cell lines grown as complex spheroids and their corresponding patient-derived xenografts (PDX). Additional PDX studies are planned for promising drug combinations from this in vitro screen. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Rene Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Siddhartha Paul, Nathan P. Coussens, Ralph E. Parchment, Joel Morris, John Wright, Naoko Takebe, Beverly A. Teicher, James H. Doroshow. Drug combination screening of Ipatasertib and Abemaciclib with other targeted agents in complex multicellular tumor spheroids from the NCI-60 and the National Cancer Institute’s Patient-Derived Models Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1881.

Phytochemical Study and Antiglioblastoma Activity Assessment of Plectranthus hadiensis (Forssk.) Schweinf. ex Sprenger var. hadiensis Stems.

Glioblastoma (GB) is the most malignant form of primary astrocytoma, accounting for more than 60% of all brain tumors in adults. Nowadays, due to the development of multidrug resistance causing relapses to the current treatments and the development of severe side effects resulting in reduced survival rates, new therapeutic approaches are needed. The genus Plectranthus belongs to the Lamiaceae family and is known to be rich in abietane-type diterpenes, which possess antitumor activity. Specifically, P. hadiensis (Forssk.) Schweinf. ex Sprenger has been documented for the use against brain tumors. Therefore, the aim of this work was to perform the bioguided isolation of compounds from the acetonic extract of P. hadiensis stems and to investigate the in vitro antiglioblastoma activity of the extract and its isolated constituents. After extraction, six fractions were obtained from the acetonic extract of P. hadiensis stems. In a preliminary biological screening, the fractions V and III showed the highest antioxidant and antimicrobial activities. None of the fractions were toxic in the Artemia salina assay. We obtained different abietane-type diterpenes such as 7α-acetoxy-6β-hydroxyroyleanone (Roy) and 6β,7β-dihydroxyroyleanone (DiRoy), which was also in agreement with the HPLC-DAD profile of the extract. Furthermore, the antiproliferative activity was assessed in a glioma tumor cell line panel by the Alamar blue assay. After 48 h treatment, Roy exerted strong antiproliferative/cytotoxic effects against tumor cells with low IC50 values among the different cell lines. Finally, we synthesized a new fluorescence derivative in this study to evaluate the biodistribution of Roy. The uptake of BODIPY-7α-acetoxy-6β-hydroxyroyleanone by GB cells was associated with increased intracellular fluorescence, supporting the antiproliferative effects of Roy. In conclusion, Roy is a promising natural compound that may serve as a lead compound for further derivatization to develop future therapeutic strategies against GB.

Synthesis and antitumor activity of novel hybrid compounds between 1,4-benzodioxane and imidazolium salts.

A series of novel hybrid compounds between 1,4-benzodioxane and imidazolium salts was designed and prepared. The compounds were evaluated in vitro against a panel of human tumor cell lines (K562, SMMC-7721, and A-549). The structure-activity relationship results demonstrated that the 2-methyl-benzimidazole or 5,6-dimethyl-benzimidazole ring and substitution of the imidazolyl-3-position with a 4-phenylphenacyl substituent were critical for promoting cytotoxic activity. Particularly, compound 25 was found to be the most potent compound with IC50 values of 1.06-8.31 μM against the three human tumor cell lines and exhibited higher selectivity to K562 and SMMC-7721 cells with IC50 values 4.5- and 4.7-fold lower than cisplatin. Moreover, compound 25 inhibited cell proliferation by inducing the G0/G1 cell cycle arrest and apoptosis in SMMC-7721 cells.

Therapeutic efficacy of antibody-drug conjugates targeting GD2-positive tumors

BackgroundBoth ganglioside GD2-targeted immunotherapy and antibody-drug conjugates (ADCs) have demonstrated clinical success as solid tumor therapies in recent years, yet no research has been carried out to develop anti-GD2 ADCs...

Design, synthesis, in silico studies, and antiproliferative evaluations of novel indolin-2-one derivatives containing 3-hydroxy-4-pyridinone fragment

Keeping in view the pharmacological properties of indolinones as promising scaffold as kinase inhibitors, herein, a novel series of 3-hydrazonoindolin-2-one derivatives bearing 3-hydroxy-4-pyridinone moiety were synthesized, studied by molecular docking, and fully characterized by spectroscopic techniques. All the prepared compounds were evaluated for their cytotoxicity attributes against a panel of tumor cell lines, including non-small cell lung cancer (A549), breast carcinoma (MCF-7), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). They displayed moderate to promising antiproliferative effects toward A549 and MCF-7 cells but remarkable results against AML and CML. Especially, compound 10k was found to be more potent against AML (EC50 = 0.69 μM) compare to the other halogen-substituted derivatives. FMS-like tyrosine kinase 3 (FLT3) is known to be expressed in AML cancer cells. The molecular docking studies demonstrated that our prepared compounds were potentially bound to AML active site through essential H-bond and other vital interactions with critical binding residues.

A tormentic acid-homopiperazine-rhodamine B conjugate of single-digit nanomolar cytotoxicity and high selectivity for several human tumor cell lines

Tormentic acid and euscaphic acid were isolated from plant material and transformed into amides holding an extra rhodamine B moiety; these compounds were screened for their cytotoxic activity employing a panel of human tumor cell lines and non-malignant fibroblasts. Prerequisites for both high cytotoxicity and tumor cell selectivity seem to be the combination of an amide (from a secondary amine but not from a primary), the use of homopiperazine rather than piperazine as a spacer together with rhodamine B (as a cationic center) and – most important-a triterpene holding a (2α, 3β) configuration of the substituents in ring A of the triterpenoid skeleton. All these features are found in tormentic acid derived compound 15; it acts as a mitocan; it is approximately 190 times more cytotoxic for ovarian carcinoma cells than for non-malignant fibroblasts, and 15 displayed an EC50 as low as 1 ​nM for several human cancer cell lines.

Synthesis and Biological Evaluation of Novel Bufalin Derivatives.

Bufalin and other cardiac steroids (CS) have been used for centuries for the treatment of congestive heart failure, arrhythmias, and other maladies. However, toxicity and the small therapeutic window of this family of steroids limit their use. Therefore, attempts to synthesize a potent, but less toxic, CS are of major importance. In the present study, two novel bufalin derivatives were synthesized and some of their pharmacological properties were characterized. The reaction of bufalin with Ishikawa’s reagent resulted in the production of two novel bufalin derivatives: bufalin 2,3-ene and bufalin 3,4-ene. The compounds were purified with TLC and HPLC and their structure was verified with UV, NMR, and MS analyses. The biological activities of these compounds were evaluated by testing their ability to inhibit the Na+, K+-ATPase activity of the brain microsomal fraction to induce cytotoxic activity against the NCI-60 human tumor cell line panel and non-cancer human cells, and to increase the force of contraction of quail embryonic heart muscle cells in culture. The two steroids exhibited biological activities similar to those of other CS in the tested experimental systems, but with reduced cytotoxicity, advocating their development as drugs for the treatment of heart failure and arrhythmias.

Cu(I) and Cu(II) Complexes Based on Lonidamine-Conjugated Ligands Designed to Promote Synergistic Antitumor Effects.

Bis(pyrazol-1-yl)- and bis(3,5-dimethylpyrazol-1-yl)-acetates were conjugated with the 2-hydroxyethylester and 2-aminoethylamide derivatives of the antineoplastic drug lonidamine to prepare Cu(I) and Cu(II) complexes that might act through synergistic mechanisms of action due to the presence of lonidamine and copper in the same chemical entity. Synchrotron radiation-based complementary techniques [X-ray photorlectron spectroscopy and near-edge X-ray absorption fine structure (NEXAFS)] were used to characterize the electronic and molecular structures of the complexes and the local structure around the copper ion (XAFS) in selected complexes. All complexes showed significant antitumor activity, proving to be more effective than the reference drug cisplatin in a panel of human tumor cell lines, and were able to overcome oxaliplatin and multidrug resistance. Noticeably, these Cu complexes appeared much more effective than cisplatin against 3D spheroids of pancreatic PSN-1 cancer cells; among these, PPh3-containing Cu(I) complex 15 appeared to be the most promising derivative. Mechanistic studies revealed that 15 induced cancer cell death by means of an apoptosis-alternative cell death.

Sclerotiamides C-H, Notoamides from a Marine Gorgonian-Derived Fungus with Cytotoxic Activities.

Bioassay-guided fractionation in association with LC-MS and NMR detection led to the isolation of six new alkaloids, sclerotiamides C-H (1-6), from the marine gorgonian-derived fungus Aspergillus sclerotiorum LZDX-33-4. Their structures were determined from extensive spectroscopic data, including ECD data and single-crystal X-ray diffraction analysis for configurational assignments. Sclerotiamides C (1) and D (2) are notoamide-type alkaloids with the incorporation of a unique 2,2-diaminopropane unit, and sclerotiamides E (3) and F (4) are unprecedented notoamide hybrids with a new coumarin unit. Sclerotiamide H (6) represents a new highly oxidized notoamide scaffold. Sclerotiamides C and F showed significant inhibition against a panel of tumor cell lines with IC50 values ranging from 1.6 to 7.9 μM. Sclerotiamide C induces apoptosis in HeLa cells by arresting the cell cycle, activating ROS production, and regulating apoptosis-related proteins in the MAPK signaling pathway. The present study extends the scaffold diversity of the notoamides and provides a potential lead for the development of a cytotoxic agent.

Targeting urothelial neoplasia using an investigational virus-like drug conjugate.

514 Background: Human papillomavirus virus-like particles (HPV VLP) preferentially target tumor cells via cell surface modified heparan-sulfate proteoglycans (HSPG). The investigational virus-like drug conjugate, belzupacap sarotalocan (AU-011), is currently in phase 2 clinical trials for treatment of primary choroidal melanoma. We previously demonstrated AU-011’s in vivo tumor acute cytotoxicity upon activation with near-infrared light (nIR), resulting in tumor-free survival for at least 100 days and protection from tumor re-challenge in the MB49 murine flank model of bladder cancer. Here we provide additional data supporting further clinical development in urothelial neoplasia as an additional oncology indication. Methods: We examined the cytotoxicity of AU-011 in a panel of six human bladder cancer cell lines in vitro and binding and distribution in human tumor biopsy samples ex vivo. Tumor distribution of AU-011 was also assessed in vivo after intravesical instillation in the orthotopic MB49 murine model. In consideration of the glycocalyx layer often found coating the interior of the bladder wall and the surface of bladder tumors, pre-treatment with hyauronidase was tested both on human biopsy samples and in the murine orthotopic model. Lastly, tumor distribution of AU-011 was evaluated when formulated with the polyamide Syn3. Results: When tested in vitro on a panel of human bladder tumor cell lines representing grades I-IV carcinomas, we found AU-011’s potency to be in the picomolar range (EC50 range 16.6 pM – 62.75 pM). We assessed the VLPs ability to bind ex vivo to a panel of human urothelial tumor biopsies and observed binding in samples representing both invasive T2 and non-invasive Ta papillary tumors. Tumor biopsy samples were pre-treated with hyaluronidase to break down the glycocalyx layer surrounding the tumor, and an increase in signal was observed in the Ta biopsy sample. Similarly, using the orthotopic MB49 murine tumor model with intravesical delivery of AU-011, we observed an enhancement of AU-011 distribution into the tumor when the bladders were pre-treated with hyaluronidase. Additionally, when formulated with Syn3, AU-011 distribution throughout the tumors was augmented without the need for hyaluronidase pre-treatment. Conclusions: We have demonstrated AU-011 targeted cytotoxicity in vitro using a panel of human bladder cancer cell lines indicating that its binding to bladder cancer cells is tumor grade and genetic mutation agnostic. Additionally, tumor binding and distribution was observed ex vivo using human bladder cancer biopsy samples and in vivo in the murine MB49 orthotopic model. Human and murine tumor distribution was improved with a pre-treatment of hyaluronidase or when formulated with Syn3. Collectively, these data support the further investigation of the use of AU-011 for the indication of uroepithelial cancer.

Abstract P5-01-10: A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes

Abstract Background: Tumor mutation loads are a strong predictor of immunotherapy benefit. This is evidenced by studies on skin, lung, and colon cancer, where well-defined processes elicit high mutation burdens and checkpoint therapies often provoke strong anti-tumor immune responses. In comparison, only a small number of breast cancer patients have responded to checkpoint therapies such as anti-PD-L1 and anti-PD-1 monoclonal antibodies. Approximately one-fourth of primary breast cancer and one-third of metastatic tumors have a significant APOBEC mutation burden defined as C-to-T and C-to-G mutations in TCA or TCT trinucleotide motifs, and many tumors have >10,000 of these APOBEC signature single base substitution mutations. However, to our knowledge, systematic immunotherapy studies focusing on this unique, hypermutated subset of breast cancers have yet to be done. Methods: To address the potential relationship between different checkpoint therapies and APOBEC mutation burden, we created a panel of >30 mammary tumor cell lines derived from fully immune-proficient C57BL/6 animals. At least 6 of these cell lines are immortalized, readily engineered using standard molecular biology procedures, and capable of re-engraftment and tumor formation in immune proficient C57BL/6 animals. Two lines were engineered to inducibly express human APOBEC3A, subjected to 1- to 20-rounds of sublethal mutagenesis, and tested systematically for checkpoint therapy responsiveness. Results: Two cell lines were engineered to inducibly express APOBEC3A or a catalytic mutant (E72A). Clones were characterized for inducible APOBEC3A expression and associated DNA damage responses. After varying rounds of mutagenesis, clones were engrafted into the subcutaneous flank or mammary fat pad of C57BL/6 animals. Immunohistochemical and flow cytometry analysis revealed a robust intratumoral CD8 T cell population including a subset with PD-1 expression. Additionally, engrafted tumor cells expressed high levels PD-L1. Experiments are ongoing and immunotherapy responses will be presented. Conclusions: We have isolated a panel of C57BL/6 mammary tumor-derived cell lines and demonstrated re-engraftment into immunocompetent animals. These cell lines constitute a platform for studying the relationship between different mutagens including APOBEC and immunotherapy responses. Citation Format: Jordan Naumann, Michael Carpenter, Prokopios Argyris, William Brown, Reuben Harris. A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-10.

Anthranilamides with quinoline and β-carboline scaffolds: design, synthesis, and biological activity.

In the present study, we report the design and synthesis of novel amide-type hybrid molecules based on anthranilic acid and quinoline or β-carboline heterocyclic scaffolds. Three types of biological screenings were performed: (i) in vitro antiproliferative screening against a panel of solid tumor and leukemia cell lines, (ii) antiviral screening against several RNA viruses, and (iii) anti-quorum sensing screening using gram-negative Chromobacterium violaceum as the reporter strain. Antiproliferative screening revealed a high activity of several compounds. Anthranilamides 12 and 13 with chloroquine core and halogenated anthranilic acid were the most active agents toward diverse cancer cell lines such as glioblastoma, pancreatic adenocarcinoma, colorectal carcinoma, lung carcinoma, acute lymphoblastic, acute myeloid, chronic myeloid leukemia, and non-Hodgkin lymphoma, but also against noncancerous cell lines. Boc-protected analogs 2 and 3 showed moderate activities against the tested cancer cells without toxic effects against noncancerous cells. A nonhalogenated quinoline derivative 10 with N-benzylanthranilic acid residue was equally active as 12 and 13 and selective toward tumor cells. Chloroquine and quinoline anthranilamides 10–13 exerted pronounced antiviral effect against human coronaviruses 229E and OC43, whereas 12 and 13 against coronavirus OC43 (EC50 values in low micromolar range; selectivity indices from 4.6 to > 10.4). Anthranilamides 14 and 16 with PQ core inhibited HIV-1 with EC50 values of 9.3 and 14.1 µM, respectively. Compound 13 displayed significant anti-quorum/biofilm effect against the quorum sensing reporter strain (IC50 of 3.7 μM) with no apparent bactericidal effect.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11030-021-10347-8.

Antibacterial and Cytotoxic Phenolic Polyketides from Two Marine-Derived Fungal Strains of Aspergillus unguis.

A chemical investigation on the EtOAc extracts from two marine-derived fungal strains of Aspergillus unguis resulted in the isolation of three previously undescribed phenolic polyketides including unguidepside C (1), aspersidone B (3), and agonodepside C (12), and their 14 known congeners. The structures of the new compounds were determined based on detailed analysis and comparison of their spectroscopic data with literature values, as well as Snatzke’s method. The new compounds (1, 3, and 12) displayed a significant anti-Gram-positive bacterial activity, with MIC values ranging from 5.3 to 22.1 µM. Additionally, the isolated compounds (1–11 and 13–16) were evaluated for their cytotoxicity against a panel of tumor cell lines. Most of them (except for 9) displayed cytotoxicity against all the tested cell lines, with IC50 values ranging from 2.5 to 46.9 µM.

Design, synthesis and study of novel indoxine derivatives

Keeping in view the pharmacological properties of indolinones as promising scaffold as kinase inhibitors, herein, a novel series of N- benzyl-4-phenyl-5-cyano-indoxine moiety were synthesized, studied by molecular docking, and fully characterized by spectroscopic techniques. All the prepared compounds were evaluated for their cytotoxicity attributes against a panel of tumor cell lines, including cardiovascular disease (ALA-564). They displayed moderate to promising antiproliferative effects toward ALA-564. The molecular docking studies demonstrated that our prepared compounds were potentially bound to 4UWC active site through essential H-bond and other vital interactions with critical binding residues.

The Strange Case: The Unsymmetric Cisplatin-Based Pt(IV) Prodrug [Pt(CH3COO)Cl2(NH3)2(OH)] Exhibits Higher Cytotoxic Activity with respect to Its Symmetric Congeners due to Carrier-Mediated Cellular Uptake.

The biological behavior of the axially unsymmetric antitumor prodrug (OC-6-44)-acetatodiamminedichloridohydroxidoplatinum(IV), 2, was deeply investigated and compared with that of analogous symmetric Pt(IV) complexes, namely, dihydroxido 1 and diacetato 3, which have a similar structure. The complexes were tested on a panel of human tumor cell lines. Complex 2 showed an anomalous higher cytotoxicity (similar to that of cisplatin) with respect to their analogues 1 and 3. Their reduction potentials, reduction kinetics, lipophilicity, and membrane affinity are compared. Cellular uptake and DNA platination of Pt(IV) complexes were deeply investigated in the sensitive A2780 human ovarian cancer cell line and in the corresponding resistant A2780cisR subline. The unexpected activity of 2 appears to be related to its peculiar cellular accumulation and not to a different rate of reduction or a different efficacy in DNA platination and/or efficiency in apoptosis induction. Although the exact mechanism of cell uptake is not fully deciphered, a series of naïve experiments indicates an energy-dependent, carrier-mediated transport: the organic cation transporters (OCTs) are the likely proteins involved.

21,24-Cyclolanostanes revisited: Structural revision and biological evaluation

Chemical fractionation of the EtOH extract of a medicinal macro fungus, Inonotus obliquus, afforded an array of lanostane-type triterpenoids (1−11) including two new ones (1 and 8). The structures of these compounds were determined on the basis of spectroscopic analyses, single crystal X-ray crystallography of 3–6 and biosynthetic considerations. With the confirmatory structural information provided by X-ray diffraction analysis in hand, several previously reported 21,24-cyclolanostanes, such as inonotsutriols A–C and (20R,21S,24S)-21,24-cyclopenta-3β,21,25-trihydroxylanosta-8-ene, were structurally corrected. In addition, the NMR data of other types of 21,24-cyclo triterpenoids were also re-examined and structural revisions were thus suggested. Compounds 2, 6 and 8 showed significant cytostatic effects against a panel of tumor cell lines with IC50 values ranging from 7.80 to 18.5 μM. Further assays established that compound 2 exerted promising in vitro anti-breast cancer potential by inhibiting the proliferation and migration of 4T1 cells.

2-Aminobenzoxazole-appended coumarins as potent and selective inhibitors of tumour-associated carbonic anhydrases

We have carried out the design, synthesis, and evaluation of a small library of 2-aminobenzoxazole-appended coumarins as novel inhibitors of tumour-related CAs IX and XII. Substituents on C-3 and/or C-4 positions of the coumarin scaffold, and on the benzoxazole moiety, together with the length of the linker connecting both units were modified to obtain useful structure-activity relationships. CA inhibition studies revealed a good selectivity towards tumour-associated CAs IX and XII (K i within the mid-nanomolar range in most of the cases) in comparison with CAs I, II, IV, and VII (K i > 10 µM); CA IX was found to be slightly more sensitive towards structural changes. Docking calculations suggested that the coumarin scaffold might act as a prodrug, binding to the CAs in its hydrolysed form, which is in turn obtained due to the esterase activity of CAs. An increase of the tether length and of the substituents steric hindrance was found to be detrimental to in vitro antiproliferative activities. Incorporation of a chlorine atom on C-3 of the coumarin moiety achieved the strongest antiproliferative agent, with activities within the low micromolar range for the panel of tumour cell lines tested.

STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

BackgroundIn breast cancer, complex interactions between tumor cells and cells within the surrounding stroma, such as macrophages, are critical for tumor growth, progression, and therapeutic response. Recent studies have highlighted the complex nature and heterogeneous populations of macrophages associated with both tumor-promoting and tumor-inhibiting phenotypes. Defining the pathways that drive macrophage function is important for understanding their complex phenotypes within the tumor microenvironment. Signal transducer and activator of transcription (STAT) transcription factors, such as STAT5, are key regulators of immune cell function. The studies described here investigate the functional contributions of STAT5 to tumor-associated macrophage function in breast cancer.MethodsInitial studies were performed using a panel of human breast cancer and mouse mammary tumor cell lines to determine the ability of tumor cell-derived factors to induce STAT5 activation in macrophages. Further studies used these models to identify soluble factors that activate STAT5 in macrophages. To delineate STAT5-specific contributions to macrophage function, a conditional model of myeloid STAT5 deletion was used for in vitro, RNA-sequencing, and in vivo studies. The effects of STAT5 deletion in macrophages on tumor cell migration and metastasis were evaluated using in vitro co-culture migration assays and an in vivo tumor cell-macrophage co-injection model.ResultsWe demonstrate here that STAT5 is robustly activated in macrophages by tumor cell-derived factors and that GM-CSF is a key cytokine stimulating this pathway. The analysis of RNA-seq studies reveals that STAT5 promotes expression of immune stimulatory genes in macrophages and that loss of STAT5 in macrophages results in increased expression of tissue remodeling factors. Finally, we demonstrate that loss of STAT5 in macrophages promotes tumor cell migration in vitro and mammary tumor metastasis in vivo.ConclusionsBreast cancer cells produce soluble factors, such as GM-CSF, that activate the STAT5 pathway in macrophages and drive expression of inflammatory factors. STAT5 deletion in myeloid cells enhances metastasis, suggesting that STAT5 activation in tumor-associated macrophages protects against tumor progression. Understanding mechanisms that drive macrophage function in the tumor microenvironment will ultimately lead to new approaches that suppress tumor-promoting functions while enhancing their anti-tumor functions.

Bivalent β-Carbolines Inhibit Colorectal Cancer Growth through Inducing Autophagy.

In this study, a series of alkyl diamine linked bivalent β-carbolines was synthesized and evaluated as antitumor agent. The results demonstrated that most compounds displayed good antiproliferative activities with IC50 value lower than 10 µM against a panel of human tumor cell lines, and compound 8 was found to be the most potent antiproliferative agent with IC50 value of 1.39, 1.96, 1.42, 1.49, 1.32, 1.96 and 1.63 µM against human breast cancer cell line (MCF-7), human adenocarcinoma cell line (769-P), human malighant melanoma cell line (A375), human ovarian cancer cell line (SK-OV-3), human colon carcinoma cell line (HCT-116), human gastric cancer cell line (BGC-823) and human esophageal squamous carcinoma cell line (Eca-109), respectively. Further investigations on mechanism of action of this class of compound demonstrated that the representative compound 8 inhibited colorectal cancer growth through inducing autophagy.