Inhibitor Of Cell Proliferation Research Articles

Identification of Macrolepiota procera extract as a novel G6PD inhibitor for the treatment of lung cancer

Tumor metabolism, an emerging hallmark of cancer, is characterized by aberrant expression of enzymes from various metabolic pathways including glycolysis and PPP (pentose phosphate pathway). Glucose 6 phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), oxidative carboxylases of PPP, have been reported to accomplish different biosynthetic and energy requirements of cancer cells. G6PD and 6PGD have been proposed as potential therapeutic targets for cancer therapy during recent years due to their overexpression in various cancers. Here, we have employed enzymatic assay based screening using in-house G6PD and 6PGD assay protocols for the identification of mushroom extracts which could inhibit G6PD or 6PGD enzymatic activity for implications in cancer therapy. For the fulfillment of the objectives of present study, nine edible mushrooms were subjected to green extraction for preparation of ethanolic extracts. 6xhis-G6PD and pET-28a-h6PGD plasmids were expressed in BL21-DE3 E. coli cells for the expression and purification of protein of interests. Using purified proteins, in house enzymatic assay protocols were established. The preliminary screening identified two extracts (Macrolepiota procera and Terfezia boudieri) as potent and selective G6PD inhibitors, while no extract was found highly active against 6PGD. Further, evaluation of anticancer potential of mushroom extracts against lung cancer cells revealed Macrolepiota procera as potential inhibitor of cancer cell proliferation with IC50 value of 6.18 μg/ml. Finally, screening of M. procera-derived compounds against G6PD via molecular docking has identified paraben, quercetin and syringic acid as virtual hit compounds possessing good binding affinity with G6PD. The result of present study provides novel findings for possible mechanism of action of M. procera extract against A549 via G6PD inhibition suggesting that M. procera might be of therapeutic interest for lung cancer treatment.

Optimization and SAR investigation of novel 2,3-dihydropyrazino[1,2-a]indole-1,4-dione derivatives as EGFR and BRAFV600E dual inhibitors with potent antiproliferative and antioxidant activities

Using a single drug to treat cancer with dual-targeting is an unusual approach when compared to other drug combinations. Dual-targeting agents were developed as a result of insufficient efficacy and drug resistance when single-targeting agents were used. As a result, the 2,3-dihydropyrazino[1,2-a]indole-1,4-dione derivatives 13–22 have been developed as dual EGFR and BRAFV600E inhibitors. The target compounds were synthesized and tested in vitro against four cancer cell lines, with compounds 15, and 19–22 demonstrating potent antiproliferative activity. In vitro studies revealed that these compounds have dual inhibitory effect on EGFR and BRAFV600E. Compounds 15, and 19–22 exhibited inhibitions of EGFR with IC50 ranging from 32 nM to 63 nM which were superior to erlotinib (IC50 = 80 ± 10 nM). Compounds 20, 21 and 22 showed promising inhibitory activity of BRAFV600E (IC50 = 55, 45 and 51 nM, respectively) and were found to be potent inhibitors of cancer cell proliferation (GI50 = 51, 35 and 44 nM, respectively). Compounds 20, 21 and 22 showed good antioxidant activity comparable to the reference Trolox. Lastly, the best active dual inhibitors were docked inside EGFR and BRAFV600E active sites to clarify their binding modes.

AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

BackgroundCurrently, multiple myeloma (MM) is still an incurable plasma cell malignancy in urgent need of novel therapeutic targets and drugs.MethodsBufalin was known as a highly toxic but effective anti-cancer compound. We used Bufalin as a probe to screen its potential targets by proteome microarray, in which AHSA1 was the unique target of Bufalin. The effects of AHSA1 on cellular proliferation and drug resistance were determined by MTT, western blot, flow cytometry, immunohistochemistry staining and xenograft model in vivo. The potential mechanisms of Bufalin and KU-177 in AHSA1/HSP90 were verified by co-immunoprecipitation, mass spectrometry, site mutation and microscale thermophoresis assay.ResultsAHSA1 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. Furthermore, AHSA1 promoted MM cell proliferation and proteasome inhibitor (PI) resistance in vitro and in vivo. Mechanism exploration indicated that AHSA1 acted as a co-chaperone of HSP90A to activate CDK6 and PSMD2, which were key regulators of MM proliferation and PI resistance respectively. Additionally, we identified AHSA1-K137 as the specific binding site of Bufalin on AHSA1, mutation of which decreased the interaction of AHSA1 with HSP90A and suppressed the function of AHSA1 on mediating CDK6 and PSMD2. Intriguingly, we discovered KU-177, an AHSA1 selective inhibitor, and found KU-177 targeting the same site as Bufalin. Bufalin and KU-177 treatments hampered the proliferation of flow MRD-positive cells in both primary MM and recurrent MM patient samples. Moreover, KU-177 abrogated the cellular proliferation and PI resistance induced by elevated AHSA1, and decreased the expression of CDK6 and PSMD2.ConclusionsWe demonstrate that AHSA1 may serve as a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma.

Multi-mycotoxins and Fungal Contaminants of Wheat from Anambra State, Nigeria

This study was carried out to determine the fungal and mycotoxins contamination of 36 Wheat (Triticum aestivium) samples purchased randomly from the seller of the agricultural produce in local markets of Anambra State, Nigeria. Results from the studies showed that two hundred and three fungal isolates consisting of 18 species of moulds and 5 species of yeasts contaminated the wheat samples at varying degrees. For moulds, Aspergillus species contaminated the samples mostly with (28) isolates followed by Penicillum species (19) isolates while Verticillium species and Cladosporium species had equal least contaminations with (3) isolates each. Among the yeast species, Candida rugosahad the highest number of contamination with (37) isolates followed by Cryptococcus laurentii (31) isolates while Candida stellatoides (9) isolates had the least contamination. Twenty-four fungal metabolites were also recovered. The concentration of trichothecene mycotoxin Deoxynivalenol (2067µg/kg), a protein synthesis and cell proliferation inhibitor in animals exceeded the maximum acceptable limits for human consumption. It can be deduced therefore that wheat circulating in Anambra State, Nigeria are variously contaminated with different xerophilic moulds and mycotoxins which can exert adverse health problems to consumers.
 Keyword: Wheat samples, fungal contaminants, multi-mycotoxins and market zones

Leptin Enhances Hepatic Fibrosis and Inflammation in a Mouse Model of Cholestasis

Leptin is an adipokine with roles in food intake and energy metabolism through its actions on neurons in the hypothalamus. The role of leptin in obesity and cardiovascular disorders is well documented. However, its influence on liver conditions such as cholestasis is poorly understood. The effects of exogenous leptin and leptin-neutralizing antibody on biliary hyperplasia, hepatic fibrosis, and inflammation in the multidrug resistance protein 2 knockout (Mdr2KO) mouse model of cholestasis were assessed by quantifying markers specific for cholangiocytes, activated hepatic stellate cells (HSCs), and cytokines. Serum and hepatic leptin were increased in Mdr2KO mice compared with FVB/NJ (FVBN) controls, and exogenous leptin enhanced biliary hyperplasia and liver fibrosis in Mdr2KO and FVBN mice. Leptin administration increased hepatic expression of C-C motif chemokine ligand 2 and IL-6 in Mdr2KO mice. In contrast, leptin-neutralizing antibody reduced intrahepatic bile duct mass and decreased HSC activation in Mdr2KO mice compared with FVBN controls. Sex-related differences were noted, with female Mdr2KO mice having more leptin than males. In cholangiocytes and LX2 cells invitro, leptin increased phosphorylated Akt and stimulated cell proliferation. Leptin receptor siRNA and inhibitors of Akt phosphorylation impaired leptin-induced cell proliferation and proinflammatory cytokines. The current data suggest that leptin is abnormally increased in cholestatic mice, and excess leptin increases ductular reaction, hepatic fibrosis, and inflammation via leptin receptor-mediated phosphorylation of Akt in cholangiocytes and HSCs.

Abstract P205: Novel 1,1-diarylethylene compounds degrade FOXM1 and selectively and potently reduce survival of high-grade serous ovarian cancer cells

Abstract Background: Forkhead box M1 (FOXM1) is a master transcriptional regulator and multifunctional oncoprotein that is widely overexpressed in ovarian cancer and associated with poor prognosis. Due to its promotion of numerous oncogenic phenotypes in ovarian cancer, FOXM1 has emerged as an attractive therapeutic target for ovarian cancer treatment. Recently, we reported that 1,1-diarylethylene compounds, NB-55, NB-73, and NB-115 (NB compounds), potently inhibit FOXM1 in breast cancer cells and mediate anti-cancer effects. Given the high molecular similarity between triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC), we characterized this new class of FOXM1 inhibitors in HGSC cells in the present study and compared their effects to other reported FOXM1 inhibitors. Methods: We conducted dose-response curves of NB compounds and other FOXM1 inhibitors in HGSC cells (CAOV3 and OVCAR4) and normal control (FT282 C11) cells. Western blot analyses probed for FOXM1, p-FOXM1, CCNB1, FOXA1, and FOXO3A in HGSC cells treated with NB compounds. We used the proteosome inhibitor MG132 to investigate the mechanism of FOXM1 inhibition by NB-73 and NB-115. We investigated FOXM1 transcriptional activity, colony formation, cell cycle, and apoptosis in HGSC cells treated with NB-73 and NB-115. Results: Dose-response curves in HGSC cells revealed IC50 values in the low nM for monensin; mid-nM for NB-73 and NB-115; high nM for thiostrepton; and low µM for NB-55, FDI-6, and n-phenylphenanthren-9-amine. RCM-1 did not affect HGSC cell proliferation. Dose-response curves in normal control fallopian tube epithelial (FTE) cells revealed IC50 values of high nM for thiostrepton and low µM for NB compounds and FDI-6. HGSC cells treated with NB compounds demonstrated decreased levels of FOXM1 and p-FOXM1 but not FOXA1 and FOXO3A. NB-73 and NB-115 decreased FOXM1 promoter activity and gene target expression but were no longer able to decrease FOXM1 levels when combined with MG132 in HGSC cells. Clonogenic assays of HGSC cells treated with NB-73 and NB-115 revealed decreased colony formation at low nM concentrations. HGSC cells treated with NB-73 and NB-115 have higher cell populations in G2/M and undergo higher rates of apoptosis compared to non-treated HGSC cells. Conclusions: NB-73 and NB-115 promote proteasomal degradation of FOXM1 and are more potent FOXM1 inhibitors of cell proliferation than previously characterized FOXM1 inhibitors in HGSC cells. NB-73 and NB-115 potently reduce HGSC survival and selectively inhibit HGSC cells as compared to normal control FTE cells. Our data encourage further assessment of NB compounds as a novel therapeutic strategy in the vast majority of HGSC that overexpress FOXM1. Support: NIH T32CA009476(CL), research grants 083(BSK) and 084(JAK) from the Breast Cancer Research Foundation, the Landfield Cancer Research Fund(BSK), NIH R03CA224339(ARK), The Marsha Rivkin Center(ARK), The Betty J. and Charles D. McKinsey Ovarian Cancer Research Fund(ARK), and The Fred & Pamela Buffett Cancer Center (NCI P30CA036727)(ARK). Citation Format: Cassie Liu, Catalina Muñoz-Trujillo, John A. Katzenellenbogen, Benita S. Katzenellenbogen, Adam R. Karpf. Novel 1,1-diarylethylene compounds degrade FOXM1 and selectively and potently reduce survival of high-grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P205.

Plasma Levels of Decorin Increased in Patients during the Progression of Breast Cancer.

Decorin (DCN), an extracellular matrix proteoglycan found in tumor surrounding tissues, is a natural inhibitor of tumor cell proliferation and invasion. We conducted a cross-sectional observation study to evaluate the association of the pathological stage with the levels of DCN in plasma or tumor surrounding tissue. Among 118 patients who underwent breast surgery, 35 were designated as carcinoma in situ (Stage 0), 39 were Stage I, and 44 were Stage II or III. The stromal expression of DCN was quantified using a semiquantitative digital image analysis after immunohistochemical staining. The concentration of DCN was evaluated with a specific ELISA. As we have previously shown, stromal DCN expression was attenuated in the patients with Stage I, whereas stromal and plasma DCN was elevated paradoxically in those with Stage II/III. The elevated plasma DCN is an independent predictive factor of Stage II/III by the multivariate logistic regression analysis. The plasma level of DCN was negatively correlated with stromal DCN expression only in patients with advanced disease (Stage II/III). The plasma level of DCN could become a useful biomarker for patients in the advanced stages. Extensive studies and further assessments are warranted for evaluating the prognostic significance and tumor characteristics to understand the clinical significances of stromal and systemic DCN.

Synthesis of Fluorinated Imidazole[4,5f][1,10]phenanthroline Derivatives as Potential Inhibitors of Liver Cancer Cell Proliferation by Inducing Apoptosis via DNA Damage.

Phenanthroline derivatives containing fluorinated imidazole ring are effective anti-neoplastic agents. Herein, a series of four fluorinated imidazole[4,5f][1,10]phenanthroline derivatives were synthesized and investigated as potential inhibitors to fight against the growth of liver cancer cells. The in vitro antitumor activity of targeted compounds have been evaluated by using MTT assay, and results showed that compound 4 (2-(2,3-difluorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) exhibited excellent inhibitory effect against the growth of various tumor cells, particularly for HepG2 cells, with IC50 value of approximately 0.29 μM. This result has been further confirmed by colony formation assay, showing that compound 4 suppressed the proliferation of HepG2 cells. Moreover, cell apoptosis (AO/PI dual staining and flow cytometry) analyses as well as comet assay showed that compound 4 may induce apoptosis of HepG2 cells through triggering DNA damage. Furthermore, the in vivo anti-tumor activity were evaluated on zebrafish bearing HepG2 cells showed that compound 4 can observably block the growth of liver cancer cells. All in together, these compounds, particularly compound 4, may be developed as a potential agent to treat liver cancer in the future.

ATR inhibition enables complete tumour regression in ALK-driven NB mouse models

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

CBIO-24. NF2 LOSS-OF-FUNCTION COOPERATES WITH HYPOXIA TO DRIVE RADIATION RESISTANCE IN GRADE 2 MENINGIOMAS

Abstract World health organization grade 2 meningiomas (G2M) are associated with an aggressive natural history characterized by frequent recurrence, resistance to radiation therapy (RT), and poor survival. We and others have shown that intratumoral necrosis is an independent predictor of disease progression in G2Ms treated with RT, suggesting that necrosis is a histopathological biomarker of radiation resistance. However, the mechanisms of radiation resistance in G2M remain unclear and treatment of radioresistant G2M is challenging. We performed genetic sequencing and histopathological analysis of 121 G2Ms and found that NF2 mutations are associated with intratumoral necrosis. To further investigate the relationship between NF2 loss-of-function, necrosis, and radiation resistance, we developed an in vitro model system using the IOMM-Lee meningioma cell line. In clonogenic survival assays, we found that NF2 knockdown under normoxia had little to no effect on radiation resistance compared to control. However, in the setting of hypoxia, NF2 knockdown significantly increased radiation resistance by maintaining cellular proliferation without impacting cell death. To understand the molecular basis of NF2 loss-of-function on radiation resistance, we performed bulk RNA sequencing of cells in our in vitro hypoxia/RT model and 10 patient G2Ms (5 necrotic and 5 non-necrotic tumors). GO and KEGG pathway analysis of the in vitro dataset revealed that NF2 deficiency triggers upregulation of pathways involved in DNA damage repair and cellular proliferation. Integration of differentially expressed genes between the in vitro NF2 loss-of-function model and necrotic patient tumors identified shared upregulation of S100A4, which promotes cell cycle progression, and downregulation of WNK2, a cell proliferation inhibitor. Together, these results suggest NF2 loss-of-function in the setting of hypoxia confers radiation resistance through a transcriptional program that promotes proliferation and DNA repair.

The Pivotal Role of Glutaminolysis in Multiple Myeloma: Novel Strategies for Target Therapies Against Myeloma

Introduction:Multiple myeloma (MM) is a uniformly fatal disorder of B cells characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Metabolic reprogramming is recognized as one of the hallmarks of cancer cells. Glutamine is the most abundant circulating amino acid in blood, glutamine metabolism through glutaminolysis may be associated with myeloma cell maintenance and survival.Materials and Methods:In this study, we investigated whether glutaminolysis was involved the proliferation in myeloma cells. We also investigated whether glutaminase (GLS) inhibitor, CB-839 could suppress myeloma cells and enhance the sensitivity of myeloma cells to histone deacetylase (HDAC) inhibition.Results:We first investigated the relationship between glutamine transporter or GLS gene expression and MM patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). Glutamine transporter genes such as SLC38A1 and SLC1A5 were increased in myeloma and plasma cell leukemia cells (GSE13591). In contrast, GLS1 expression was not changed. We next investigated the glutaminolysis in myeloma cells. Deprivation of glutamine in culture medium revealed that cellular growth inhibition and cell cycle arrest at G0/G1 phase. Gene expression of AURKA (aurora kinase A), AURKB (aurora kinase B), HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A Member 1) and CCNB1 (cyclin B1) were reduced from the public microarray datasets (GSE59931) and protein expressions were also reduced by immunoblot analysis. We next evaluated the effect of GLS inhibitor, CB-839. 72 h treatment of MM cells were inhibited by CB-839 in a dose dependent manner. Cellular cytotoxicity was also increased. Glutamine is converted by GLS into glutamate and alpha-ketoglutarate (α-KG), and related nicotinamide adenine dinucleotide phosphate (NADP) production. Intracellular α-KG and NADPH were reduced by CB-839. As metabolites are the substrates used to generate chromatin modification including acetylation of histone, we investigated HDAC inhibitor, panobinostat in myeloma cells. 72 h treatment of MM cells were inhibited by panobinostat and histone acetylation was increased. Combined treatment with panobinostat and CB-839 caused more cytotoxicity than each drug alone. Panobinostat and CB-839 also inhibited bortezomib resistant cells. Caspase 3/7 activity and cellular cytotoxicity were also increased. Proteasomal activity was reduced. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by panobinostat and CB-839 on myeloma cells. Immunoblot analysis revealed that protein expression of aurora kinase A, aurora kinase B, HSP90 and cyclin B1 were reduced, and cleaved caspase 3 and γ- H2AX were increased by panobinostat and CB-839 treatment. GLS shRNA transfectant cells were inhibited cellular proliferation and sub-G1 phase was increased by cell cycle analysis. GLS shRNA transfectant cells were increased the sensitivity of panobinostat compared to control cells.Conclusion:The glutaminolysis is involved myeloma cell proliferation and GLS inhibitor is effective to myeloma cells and enhance cytotoxic effects of HDAC inhibitors. We also provide the promising clinical relevance as a candidate drug for treatment of myeloma patients. DisclosuresNo relevant conflicts of interest to declare.

AdipoRon negatively regulates proliferation and migration of ARPE-19 human retinal pigment epithelial cells

Adiponectin is an adipokine playing important roles in metabolic, inflammatory and proliferative processes. At the time of surgery for rhegmatogenous retinal detachment, an altered expression of adipokines has been associated with the development of future proliferative vitreoretinopathy (PVR); this evidence as well as the presence of adiponectin receptors in ocular tissues and cell lines suggests a role of adiponectin in the physio-pathology of ocular conditions. Here, we investigated the effects of AdipoRon, an adiponectin agonist, on ARPE-19, a human retinal pigment epithelial cell line after confirming the expression of AdipoR1, AdipoR2, T-cadherin receptors. We evaluated the effects of AdipoRon in terms of vitality, survival, and migration; furthermore, we investigated the potential effects of AdipoRon on the inflammatory state of ARPE-19 cells analysing the levels of IL-10, VEGF, MCP-1 and IL-6 cytokines. Our findings indicated that AdipoRon, in a time and dose-dependent manner, reduces cell proliferation, migration, and colony formation of ARPE-19 cells. On the contrary, AdipoRon administration does not affect the expression of the tested cytokines. In conclusion, our results indicated that AdipoRon, may constitute an endogenous inhibitor of retinal pigment epithelial cell proliferation and migration, both processes deeply involved in development of PVR. Since PVR are characterized by an aberrant growth, migration and dedifferentiation of retinal pigment epithelial cells, our data contribute to open new fields of research to develop innovative therapeutic targets. Further studies are needed to clarify the effects of AdipoRon and of other small-molecule adiponectin analogs on retinal epithelium to clarify the functional role of adiponectin.

Decoupling tumor cell metastasis from growth by cellular pilot protein TNFAIP8.

Cancer metastasis accounts for nearly 90% of all cancer deaths. Metastatic cancer progression requires both cancer cell migration to the site of the metastasis and subsequent proliferation after colonization. However, it has long been recognized that cancer cell migration and proliferation can be uncoupled; but the mechanism underlying this paradox is not well understood. Here we report that TNFAIP8 (tumor necrosis factor-α-induced protein 8), a "professional" transfer protein of phosphoinositide second messengers, promotes cancer cell migration or metastasis but inhibits its proliferation or cancer growth. TNFAIP8-deficient mice developed larger tumors, but TNFAIP8-deficient tumor cells completely lost their ability to migrate toward chemoattractants and were defective in colonizing lung tissues as compared to wild-type counterparts. Mechanistically, TNFAIP8 served as a cellular "pilot" of tumor cell migration by locally amplifying PI3K-AKT and Rac signals on the cell membrane facing chemoattractant; at the same time, TNFAIP8 also acted as a global inhibitor of tumor cell growth and proliferation by regulating Hippo signaling pathway. These findings help explain the migration-proliferation paradox of cancer cells that characterizes many cancers.

Nanoparticle-eluting stents for coronary intervention: formulation, characterization, and in vitro evaluation.

Coronary artery disease (CAD) is currently a leading cause of death worldwide. In the history of percutaneous coronary intervention for the treatment of CAD, a drug-eluting stent (DES) is recognized as a revolutionary technology that has the unique ability to significantly reduce restenosis and provide both mechanical and biological solutions simultaneously to the target lesion. The aim of the research work was to design and fabricate DES coated with a nanoparticulate drug formulation. Sirolimus, an inhibitor of the smooth muscle cell (SMC) proliferation and migration, was encapsulated in polymeric nanoparticles (NPs). The NP formulation was characterized for various physicochemical parameters. Cell viability and cell uptake studies were performed using human coronary artery smooth muscle cells (HCASMCs). The developed NP formulation showed enhanced efficacy compared to plain drug solution and exhibited time-dependent uptake into the HCASMCs. The developed NP formulation was coated on the Flexinnium™ ultra-thin cobalt-chromium alloy coronary stent platform. The NP-coated stents were characterized for morphology and residual solvent analysis. In vitro drug release was also evaluated. Ex vivo arterial permeation was carried out to evaluate the NP uptake from the surface of the stents. The characterization studies together corroborated that the developed NP coated stent can be a promising replacement of the current DESs.

Identification of a novel inhibitor of liver cancer cell invasion and proliferation through regulation of Akt and Twist1

When primary cancer faces limited oxygen and nutrient supply, it undergoes an epithelial–mesenchymal transition, which increases cancer cell motility and invasiveness. The migratory and invasive cancer cells often exert aggressive cancer development or even cancer metastasis. In this study, we investigated a novel compound, 3-acetyl-5,8-dichloro-2-((2,4-dichlorophenyl)amino)quinolin-4(1H)-one (ADQ), that showed significant suppression of wound healing and cellular invasion. This compound also inhibited anchorage-independent cell growth, multicellular tumor spheroid survival/invasion, and metalloprotease activities. The anti-proliferative effects of ADQ were mediated by inhibition of the Akt pathway. In addition, ADQ reduced the expression of mesenchymal markers of cancer cells, which was associated with the suppressed expression of Twist1. In conclusion, ADQ successfully suppressed carcinogenic activity by inhibiting the Akt signaling pathway and Twist1, which suggests that ADQ may be an efficient candidate for cancer drug development.

Proteomics study of Mycoplasma pneumoniae pneumonia reveals the Fc fragment of the IgG-binding protein as a serum biomarker and implicates potential therapeutic targets.

Macrolide and corticosteroid resistance has been reported in patients with Mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult to achieve through antibiotic treatment in sensitive patients with severe MPP (SMPP). SMPP in children might progress to airway remodeling and even bronchiolitis/bronchitis obliterans. Therefore, identifying serum biomarkers that indicate MPP progression and exploring new targeted drugs for SMPP treatment require urgency. In this study, serum samples were collected from patients with general MPP (GMPP) and SMPP to conduct proteomics profiling. The Fc fragment of the IgG-binding protein (FCGBP) was identified as the most promising indicator of SMPP. Biological enrichment analysis indicated uncontrolled inflammation in SMPP. ELISA results proved that the FCGBP level in patients with SMPP was substantially higher than that in patients with GMPP. Furthermore, the FCGBP levels showed a decreasing trend in patients with GMPP but the opposite trend in patients with SMPP during disease progression. Connectivity map analyses identified 25 possible targeted drugs for SMPP treatment. Among them, a mechanistic target of rapamycin kinase (mTOR) inhibitor, which is a macrolide compound and a cell proliferation inhibitor, was the most promising candidate for targeting SMPP. To our knowledge, this study was the first proteomics-based characterization of patients with SMPP and GMPP.

Abstract LB003: Fragment based discovery of MRTX9768, a synthetic lethal-based inhibitor designed to bind the PRMT5-MTA complex and selectively target MTAP/CDKN2A-deleted tumors

Abstract The MTAP gene is proximal to and co-deleted in nearly all CDKN2A-deleted cancers. This genetic alteration is present in an estimated 9% of all cancers and is especially prevalent in cancers with high unmet medical need (e.g. mesothelioma (32%), pancreatic (22%), lung squamous (20%)). Multiple independent research teams have demonstrated that tumor cell lines with homozygous MTAP deletions are hypersensitive to shRNA-mediated knock down of PRMT5. MTAP is required for the methionine salvage pathway and MTAP-del cells accumulate MTA, an inhibitory co-factor which competes for binding to the co-factor binding site of PRMT5 with the activating co-factor SAM. PRMT5 is a methyltransferase that adds symmetric dimethylarginine (SDMA) modification to proteins and is essential for mammalian cell survival. A small molecule that selectively binds and stabilizes the catalytically inactive PRMT5•MTA complex may represent a synthetic lethal-based precision medicine for the treatment of MTAP/CDKN2A—del tumors. Notably, 1st generation PRMT5 small molecule inhibitors do not target MTA-complexed PRMT5 and do not exhibit selective inhibition of MTAP-del cancer cells. Here we report a new series of compounds discovered via a fragment-based approach that selectively bind to the PRMT5•MTA complex. A fragment hit was identified in an SPR binding assay with PRMT5•MTA (KD 18 μM). The binding mode was determined by X-ray crystallography and revealed that the fragment makes productive interactions with K333, F327, E435, E444, E435, and W579 of PRMT5 as well as with the co-liganded MTA. Fragment growing aided by structure-based design identified a key interaction with the L312 backbone N-H that enhances binding to PRMT5•MTA (MRTX4646, SPR KD 57 nM). Further exploration highlighted an interaction with the F580 backbone N-H as important for cellular activity and selectivity. This interaction with F580 was illustrated by MRTX7512 which exhibits an IC50 value of 633 nM for inhibition of SDMA in engineered HCT116 MTAP-del cells and demonstrates 15-fold selectivity compared with HCT116 MTAP-WT cells (IC50 9763 nM). Further optimization of cellular potency and pharmacokinetic properties identified MRTX9768, a potent inhibitor of SDMA and cell proliferation in HCT116 MTAP-del cells (SDMA IC50 3 nM; prolif. IC50 11 nM) with marked selectivity over HCT116 MTAP-WT cells (SDMA IC50 544 nM; prolif. IC50 861 nM). In xenograft studies, oral administration of MRTX9768 demonstrates dose-dependent inhibition of SDMA in MTAP-del tumors, with less SDMA modulation observed in bone marrow. In summary, we have used a fragment-based approach to discover a new class of orally active PRMT5•MTA inhibitors that demonstrate selective antitumor activity in MTAP-del tumor cells while sparing MTAP-WT cells. Citation Format: Christopher R. Smith, Svitlana Kulyk, J. D. Lawson, Lars D. Engstrom, Ruth Aranda, David M. Briere, Robin Gunn, Krystal Moya, Lisa Rahbaek, Laura Waters, Anthony Ivetac, James G. Christensen, Peter Olson, Matthew A. Marx. Fragment based discovery of MRTX9768, a synthetic lethal-based inhibitor designed to bind the PRMT5-MTA complex and selectively target MTAP/CDKN2A-deleted tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB003.

Adrenocortical Carcinoma Steroid Profiles: In Silico Pan-Cancer Analysis of TCGA Data Uncovers Immunotherapy Targets for Potential Improved Outcomes.

Despite progress in understanding the biology of adrenocortical carcinoma (ACC), treatment options have not dramatically changed in the last three decades, nor have we learned how to avoid some of its long-term side effects. Our goal was to improve the understanding of immune pathways that may include druggable targets to enhance immune responses of patients with ACC, focusing on immune evasion and the activation of immune cells against ACC. Our strategy was aimed at improving insight regarding gene expression without steroid interference. Using approaches based on high and low steroid phenotypes (HSP and LSP, respectively), we characterized immune pathways using The Cancer Genome Atlas (TCGA) ACC cohort data. Although previous studies have suggested that patients with ACC receive minimal benefit from immunotherapy, high expression of immune modulators was noted in patients with LSP, suggesting the activation of these biomarkers may be an important adjuvant therapy target after clearance of excess glucocorticoids. In addition, patients with LSP ACC had higher immune cell infiltration than patients with HSP ACC and other cancer subtypes. Our findings can be summarized as follows (1): we confirmed and improved the definition of two immune response pathways to ACC (HSP and LSP) based on in silico transcriptome analysis (2), we demonstrated the steroid profile should be considered, otherwise analyses of ACC immune characteristics can generate confounding results (3), among the overexpressed immunotherapy targets, we demonstrated that LSP was rich in PDCD1LG2 (PD-L2) and both HSP and LSP overexpressed CD276 (B7-H3), which was associated with resistance to anti-PD1 therapy and may have accounted for the modest results of previous clinical trials, and (4) identification of patients with LSP or HSP ACC can be used to help determine whether immunotherapy should be used. In conclusion, we highlighted the differences between LSP and HSP, drawing attention to potential therapeutic targets (CD276, PDCD1, and PDCD1LG2). Treatments to reduce immune evasion, as well as the use of other natural and pharmacological immune activators, should include prior pharmacological inhibition of steroidogenesis. Attempts to combine these with tumor cell proliferation inhibitors, if they do not affect cells of the immune system, may produce interesting results.

FC 038CRESCENTS DERIVE FROM SINGLE PODOCYTE PROGENITORS AND A DRUG ENHANCING THEIR DIFFERENTIATION ATTENUATES RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS

Abstract Background and Aims Rapidly progressive glomerulonephritis (RPGN) encompasses a group of diverse disorders characterized by the presence of massive hyperplasia of parietal epithelial cells (PEC) as the main histopathological lesion at kidney biopsy. It is associated with a rapid decline in kidney function referred to altogether as rapidly progressive glomerulonephritis. Typically, crescent formation is the consequence of diverse upstream pathomechanisms involving the specific activation of PEC. PEC normally reside peacefully along Bowman capsule and represent in part renal progenitor cells (RPC). Previous studies observed RPC markers in crescents from patients with different types of glomerulonephritis. Similarities between stem cell niches of bone marrow and kidney, prompted us to hypothesized that crescents result from monoclonal expansion of a single RPC clone conceptually similar to monoclonal diseases originating from hematopoietic stem cells. According to this analogy, we further hypothesized that drugs known to cure monoclonal disease of the hematopoietic stem cells by enforcing their terminal differentiation could also attenuate crescentic glomerulonephritis. Method To address this hypothesis, we established a RPGN disease model in a conditional transgenic mouse based on the mT/mG and the Confetti reporter that allows lineage tracing and clonal analysis of RPCs. Animals were treated with known pharmacological inhibitors of clonal stem cell proliferation in myeloproliferative disorders. Crescentic lesions were characterized by super-resolution STED microscopy. Finally, we employed single cell RNA sequencing of human renal progenitor cultures to identify the immature progenitor subset-generating crescent in human to identify putative new biomarker(s) of RPNG to validate in biopsy of patients. Results We observed that the crescentic lesions originated from the clonal expansion of single RPC, thus suggesting a clonal stem cell disorder. Therefore, we administrated a series of drugs known to ameliorates myeloproliferative neoplasms to our RPGN mouse model as potential therapeutic agents. In particular, treatment with one of the compounds induced a reduction in both proteinuria and crescent formation. 3D confocal microscopy and STED super-resolution imaging of glomeruli showed that this compound turned the uncontrolled hyperplasia of a specific immature PEC subset into a controlled differentiation into new podocytes thereby restoring the injured glomerular filtration barrier. Single cell RNA sequencing of human renal progenitor cultures identified a new marker of the crescent-generating progenitor cells. Expression of this marker in biopsies of patients with rapidly progressive glomerulonephritis associated with progression toward end stage kidney disease. Treatment of human PEC with the drug that in in vivo experiments showed a therapeutic effect on RPGN reduced proliferation of the immature progenitor subset promoting their differentiation into podocytes. Conclusion These results demonstrate that glomerular hyperplastic lesions derive from clonal amplification of a RPC subset and that shifting proliferation to podocyte differentiation reverses crescent formation and improves clinical outcome.

Trans-Cinnamic acid, but not p-coumaric acid or methyl cinnamate, induces fibroblast migration through PKA- and p38-MAPK signalling pathways

AimHydroxycinnamic acids their derivatives have various pharmacological properties. The hydroxycinnamic acid derivatives, methyl cinnamate, trans-cinnamic, and p-coumaric acids have been the object of study in the treatment of skin wounds. However, it is unclear whether these derivatives exert a direct beneficial effect on fibroblast function. In this study, we evaluated the effects of methyl cinnamate, trans-cinnamic, and p-coumaric acids on fibroblast migration in vitro. Materials and methodsNIH 3T3 and L929 fibroblast cell lines were exposed to each drug at several concentrations and the effect on cell viability, cell cycle, and extracellular matrix production were assessed by MTT assay, flow cytometry, and immunofluorescence staining, respectively. The effect on cell migration was examined using scratch assay. ResultsThe results showed that hydroxycinnamic acid derivatives not affect cell viability, but increase fibroblast migration in the in vitro scratch-wound healing assay. They also induced an increase in S and G2/M phases accompanied by a decrease in the G0/G1 phase of the cell cycle. The cell proliferation inhibitor mitomycin C abolished the effect induced by p-coumaric acid and methyl cinnamate, indicating that only the trans-cinnamic acid stimulated migration. A transwell migration assay confirmed that trans-cinnamic acid-treated fibroblasts exhibited increased migration compared with untreated cells. trans-Cinnamic acid-induced fibroblast migration was decreased by PKA inhibitor and p38-MAPK inhibitor but not by JNK inhibitor. Additionally, trans-cinnamic acid-treated fibroblasts showed an increase in the production of laminin and collagen type I. ConclusionOur study showed that trans-cinnamic acid improves fibroblast migration and modulates extracellular matrix synthesis, indicating its potential for accelerating the healing process.