Significant Reduction In Cell Growth Research Articles

Abstract A036: Macropinocytosis mediates metabolic resistance to loss of glutamine transport in triple-negative breast cancer

Abstract Introduction Although a nonessential amino acid in normal cells, the demand for glutamine is dramatically increased throughout malignant transformation, supporting a range of metabolic processes including mitochondrial ATP production, protein synthesis, purine and pyrimidine biosynthesis. We previously shown that triple-negative breast cancer (TNBC) cells rely on glutamine uptake by the amino acid transporter ASCT2 to sustain their unique glutamine metabolism, thereby supporting in vitro growth and in vivo tumour formation. However, it is known that TNBC cells can also utilise non-transporter mediated nutrient uptake facilitated by processes such as macropinocytosis. Methods We examined proliferation of human breast cancer cell lines after ASCT2 CRISPR/Cas9 knockout (clonal and polyclonal populations) and shRNA knockdown. Proteomics and mRNAseq analysis further examined cellular and adaptive changes to ASCT2 knockout. Cellular changes were further analysed by western blotting, with macropinocytosis examined using 70kDa dextran-FITC uptake. Metabolic changes were assessed using targeted metabolomics approaches including 13C-labelled substrate tracing and liquid chromatography coupled tandem-mass spectrometry (LC-MS/MS) to determine intracellular levels of key tricarboxylic acid (TCA) cycle intermediates, glycolytic metabolites, fatty acid precursors, nucleotides, and amino acids in human TNBC cell lines in vitro. Results Despite our previous data showing a significant reduction in cell growth after ASCT2 knockdown, ASCT2 knockout was well-tolerated by both TNBC and Luminal A breast cancer cell lines, with proliferation rates similar to non-targeted CRISPR/Cas9 control cells. This adaptation to ASCT2 knockout was not due to the high glutamine levels present in culture media, as the knockout cells could be cloned in media containing either standard 2 mM glutamine or physiological 0.5 mM glutamine. Previous data have shown that TNBC cell lines can undergo constitutive macropinocytosis, and that this can be enhanced when cells are cultured in low nutrient conditions. Indeed, not only did the TNBC cell line HCC1806 undergo constitutive macropinocytosis, the amount of macropinocytosis was significantly increased (5-10 fold) in 5 separate ASCT2 knockout clones tested. By comparison, the ASCT2 shRNA knockdown cell line, which are published to have a significant proliferation deficit, showed a modest 2-fold increase in macropinocytosis, which may explain why they maintain a growth phenotype compared to the knockout lines. Despite in-depth analysis of gene and protein levels by mRNAseq and proteomics, ASCT2 knockout cells showed only a few significant changes in known macropinocytotic genes. There was, however, a substantial upregulation of Ser473-Akt phosphorylation which may drive the adaptive macropinocytosis in TNBC. Conclusion These data suggest that the constitutive macropinocytosis present in TNBC cell lines provides a novel resistance mechanism to strategies targeting glutamine uptake alone. Citation Format: Kanu Wahi, Natasha Freidman, Qian Wang, Yi Fang Guan, Jeff Holst. Macropinocytosis mediates metabolic resistance to loss of glutamine transport in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A036.

Effect of Polyunsaturated Fatty Acids on Temozolomide Drug-Sensitive and Drug-Resistant Glioblastoma Cells.

Glioblastomas (GBMs) are notoriously difficult to treat, and the development of multiple drug resistance (MDR) is common during the course of the disease. The polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have been reported to improve MDR in several tumors including breast, bladder, and leukaemia. However, the effects of PUFAs on GBM cell MDR are poorly understood. The present study investigated the effects of PUFAs on cellular responses to temozolomide (TMZ) in U87MG cells and the TMZ-resistant (TMZR) cells derived from U87MG. Cells were treated with PUFAs in the absence or presence of TMZ and dose-response, viable cell counting, gene expression, Western blotting, flow cytometry, gas chromatography-mass spectrometry (GCMS), and drug efflux studies were performed. The development of TMZ resistance caused an increase in ABC transporter ABCB1 and ABCC1 expression. GLA-, EPA-, and DHA-treated cells had altered fatty acid composition and accumulated lipid droplets in the cytoplasm. The most significant reduction in cell growth was seen for the U87MG and TMZR cells in the presence of EPA. GLA and EPA caused more significant effects on ABC transporter expression than DHA. GLA and EPA in combination with TMZ caused significant reductions in rhodamine 123 efflux from U87MG cells but not from TMZR cells. Overall, these findings support the notion that PUFAs can modulate ABC transporters in GBM cells.

Pre-Clinical Investigation of a Novel Small Molecule Inhibitor Targeting YBX1 in AML

Recently, our group identified the pleiotropic DNA and RNA binding protein YBX1 as a specific dependency and therapeutic target in acute myeloid leukemia (AML) that is essential for stabilizing oncogenic transcripts and thereby maintains oncogenic protein expression (Perner et al., 2021). Selective pharmacologic targeting of YBX1 in AML, however, has not been achieved so far. Here, we investigate the functional consequences of a novel small molecule inhibitor (SU056) of YBX1 in AML. We examined the viability of several human AML cell lines (MOLM13, HL60, EOL1, THP1, Kasumi1, ML2, Mv4;11, OCI-AML3, NOMO1) upon SU056 treatment and found significant reduction of cell growth in a concentration dependent manner. Inhibition of cell growth could be mainly attributed to impaired proliferative capacity at nanomolar concentrations. Pharmacologic inhibition of YBX1 resulted in impaired colony formation (mean colony number DMSO: 231.3±13.88 vs. SU056 500nM: 154.7±8.29; p<0.0001), cell cycle arrest (cells in S phase: DMSO: 37.3±2.4 vs. SU056 500nM: 27.2±1.67; p=0.0127) and induction of myeloid differentiation. Consistently, in a xenograft model of human AML, SU056 treatment resulted in prolonged disease latency (MOLM13 diluent: 62 days OS vs. SU056: not reached, p=0.0013; OCI-AML3 diluent: 79 days OS vs. not reached, p=0.0001) and significant reduction of disease penetrance (MOLM13 diluent: 100% vs. SU056: 24,9%; OCI-AML3 diluent: 100% vs. SU056: 9,1%). Consistently, percentage of SU056 treated human leukemic cells in the bone marrow of recipient mice was decreased for both, MOLM13 (mean diluent: 41,3% vs. SU056: 7,6%) and OCI-AML3 (mean diluent: 28,9% vs. SU056: 2,9%). Likewise, pharmacologic inhibition of YBX1 reduced colony formation and total cell numbers of primary human AML cells in a dose-dependent manner. To be clinically relevant, such treatment must have a suitable therapeutic index between human hematopoietic stem- and progenitor cells (HSPCs) and their malignant counterpart. This is particularly important, considering the role of YBX1 in mRNA splicing and processing. Of note, pharmacologic inhibition of YBX1 did not perturb colony formation of normal HSPCs derived from apheresis donors. These findings were confirmed by investigating primary human leukemic cells in a patient-derived xenograft (PDX) model of AML. We aimed to assess whether loss in leukemia cell competition induced by pharmacologic targeting of YBX1 is sufficient for normal hematopoietic cells to outcompete leukemic cells in vivo. 1x106 normal human HSPCs were transplanted along with 2x104 leukemic cells to assess for efficacy of SU056-treatment versus diluent control. In vivo treatment with SU056 (20mg/kg, i.p.) for 3 weeks resulted in a significant decrease in the AML blast number in the PB (mean blast %: diluent: 38.5%; SU056: 2.25%; p<0.0001). This resulted in increased survival of SU056 treated recipients (OS diluent: 83 days vs. SU056: not reached). To confirm the proposed mechanism by which SU056 impairs leukemia growth we performed global transcriptome and proteome analysis following treatment with SU056 (500nM, 96h). Proteome profiling revealed a total of 2641 deregulated proteins. Gene-ontology analysis indicated deregulation of YBX1 targets and proteins associated with cell cycle, DNA repair, mRNA splicing/processing, and transcriptional regulation. Of note, overlap between transcriptional regulation (as determined by mRNA-Seq) and changes in proteome abundance revealed less than 5% target genes/proteins, indicating that SU056 may in fact interfere with binding of oncogenic mRNAs to YBX1 and thereby may reduce translation of oncogenic transcripts. In order to define functional consequences of pharmacologic YBX1 inhibition and explore potential vulnerabilities of drug combinations, we performed a genome-wide CRISPR/Cas9 screen in human AML cells treated with SU056 versus diluent control. Validation experiments are currently ongoing. Taken together, our data confirm the therapeutic potential to target YBX1 using the novel small molecule SU056. Given its broad applicability across genetic subtypes of AML and lack of toxicity on normal HSPCs, SU056 as a single agent or in combination with established targeted therapies may efficiently help to target and outcompete leukemic cells. Experiments to assess for potential synergy with already established targeted therapies will be presented.

Counteractions of a Novel Hydroalcoholic Extract from Lens Culinaria against the Dexamethasone-Induced Osteoblast Loss of Native Murine Cells.

The cytoprotective effects of a novel hydroalcoholic extract (0.01–5 mg/mL) from Lens culinaria (Terre di Altamura Srl) were investigated within murine native skeletal muscle fibers, bone marrow cells, and osteoblasts, and in cell lines treated with the apoptotic agent staurosporine (2.14 × 10−6 M), the alkylating drug cisplatin (10−4 M), the topoisomerase I inhibitor irinotecan (10−4 M), the antimitotic pro-oxidant doxorubicin (10−6 M), and the immunosuppressant dexamethasone (2 × 10−6 M). An amount of 10g of plant material was used to obtain a 70% ethanol/water product, following two-step extraction, evaporation, lyophilization, and storage at −20 °C. For the murine osteoblasts, doxorubicin reduced survival by −65%, dexamethasone by −32% and −60% after 24 and 48 h of incubation time, respectively. The extract was effective in preventing the osteoblast count-reduction induced by dexamethasone; it was also effective at preventing the inhibition of mineralization induced by dexamethasone. Doxorubicin and cisplatin caused a significant reduction in cell growth by −77% for bone marrow cells, −43% for irinotecan, and −60% for dexamethasone, but there was no evidence for the cytoprotective effects of the extract in these cells. Staurosporine and doxorubicin caused a fiber death rate of >−40% after 18 and 24 h of incubation, yet the extract was not effective at preventing these effects. The extract was effective in preventing the staurosporine-induced reduction of HEK293 proliferation and colony formation in the crystal violet DNA staining and the clonogenic assays. It was also effective for the cisplatin-induced reduction in HEK293 cell proliferation. The extract, however, failed to protect the SHSY5Y neurons against cisplatin and irinotecan-induced cytotoxicity. A UV/VIS spectroscopy analysis showed three peaks at the wavelengths of 350, 260, and 190 nm, which correspond to flavonoids, proanthocyanins, salicylates, and AA, constituting the extract. These data suggest the possible development of this extract for use against dexamethasone-induced bone loss and renal chemotherapy-induced damage.

Clusterin mRNA silencing reduces cell proliferation, inhibits cell migration, and increases CCL5 expression in SW480, SW620, and Caco2 cells

Abstract Objectives This study aimed to investigate the effects of specific gene silencing in colorectal cancer cells. Clusterin protein was found in the serum samples of colorectal cancer patients infected with Schistosoma mansoni previously. Methods For this reason, silencing clusterin mRNA in colorectal cancer cells was first performed to study the cytotoxic effect by lactate dehydrogenase assay. Next, propidium iodide staining and flow cytometry were performed to investigate the cell cycle profile in clusterin-silenced cells. A wound-healing assay was also used to examine the migration rate of clusterin-silenced cells. The mRNA expression of cell proliferation- and migration-related genes was then assessed by real-time PCR. Results Clusterin mRNA silencing caused a significant reduction in cell growth but induced no cell cycle arrest or potential apoptosis in all cells. It was found in this study that cell migration rate was inhibited in clusterin-silenced cells. Surprisingly, significantly induced chemokine (C–C motif) ligand 5 (CCL5) mRNA expression was detected in clusterin-silenced Caco2, which indicated that the cell proliferation and migration of clusterin-silenced Caco2 were likely associated with CCL5 mRNA expression. Conclusions Clusterin may be a potential target for regulation, staging, surveillance, and developing a cost-effective therapeutic agent for treating parasite-infected Caco2 type of colorectal cancer patients.

Anticarcinogenic Efficacy of Fucoxanthin on HepG2 Cell Lines

Introduction: Hepatocellular Carcinoma (HCC) is considered one of the deadliest cancers which grow rapidly worldwide. Fucoxanthin can be acquired from edible brown seaweeds, reported that fucoxanthin has numerous physiological functions and biological abilities, and various medicinal properties. Aim: To evaluate the anti-carcinogenic efficiency of fucoxanthin against the Human Hepatoma Cell Line (HepG2). Materials and Methods: This in-vitro examination and research was carried from June 2018 to August 2018, at Alagappa University, Karaikudi, Tamil Nadu, India. Fucoxanthin at different concentrations was taken to demonstrate its antiproliferative potential and its cytotoxic effect. In this present examination, based on the outcome of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide (MTT) assay, 80 µM and 100µM were chosen for further in-vitro studies. It was noted that remarkable leakage of Lactate Dehydrogenase (LDH) and a noteworthy decrease in the Glutathione (GSH) content of 80µ M and 100µM of fucoxanthin treated HepG2 cells. Under light and fluorescence microscopic examination, there was a significant reduction in cell growth and cell proliferation in the fucoxanthin-treated HepG2 cells. Data were presented as the mean±standard deviation. The one-way analysis of variance (ANOVA) and Tukey’s multiple comparison method were used to find the level of significance. Results: The human HepG2 to be studied and analysed were divided into group I: control group; group II: comprising those cells treated with fucoxanthin in concentration of 80 µM; group III: comprising those cells treated with fucoxanthine in concentration of 100 µM. The results of the MTT assay put forward that fucoxanthin has the cytotoxic and anti-proliferative potential against HepG2 further fucoxanthin significantly alter the marker enzymes level (p-value &lt;0.05). The therapeutic efficiency of fucoxanthin might be due to the antioxidant effect of the bioactive compound. In this present investigation, the results of in-vitro studies, fucoxanthin strongly put forward that it has potential anticarcinogenic efficacy against the hepatic tumour (p-value &lt;0.05). Conclusion: The results of this analysis undoubtedly symbolise that fucoxanthin has anti-oxidant activity and anti-carcinogenic efficacy against primary liver cancer HCC. Due to their therapeutic efficacy, may be considered as an excellent candidate against HCC.

Secretome of adipose derived stem cells induced apoptosis in anaplastic thyroid carcinoma C-643 cells

Introduction: Previous studies have proven that secretory materials of mesenchymal stem cells (MSCs) (secretome) have remedial properties. Objectives: The main goal of this study was to evaluate the effects of secretome of adipose-derived MSCs on growth and apoptosis in anaplastic thyroid carcinoma (ATC) C-643 cells. Materials and Methods: Initially, thyroid carcinoma cells were exposed to the 25 and 50 μg/mL adipose-derived stem cells (ADSCs) secretome for 24 and 48 hours, to evaluate the proliferation and cytotoxicity of the C-643 cells, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test and colony assay was conducted. Acridine orange/ethidium bromide (AO/EB) staining was conducted to evaluate the apoptosis. The expression level of apoptosis-associated genes was determined by real-time polymerase chain reaction (PCR) technique. Results: Cell viability and colony numbers in groups exposed to secretome were significantly lower than the control group. The amount of apoptosis-related genes (Bax/Bcl-2, P53, caspase-3 and caspase-8) expression in secretome-treated groups was more than the control group. Conclusion: The data revealed that ADSCs secretome caused the significant reduction of cell growth and induced apoptosis in C-643 cells.

Batch fermentation of succinic acid from cheese whey by Actinobacillus succinogenes under variant medium composition.

Bio-based succinic acid production has attracted global attention since its consideration as a potential replacement to petroleum-based platform chemicals. This study used three different CO2 sources, namely NaHCO3, K2CO3 and MgCO3 for fermentation of succinic acid (SA) by Actinobacillus succinogenes under three distinct substrate conditions i.e. lactose, whey and whey devoid of any supplements. Batch experiments were performed in both anaerobic flasks and 5L benchtop fermenter. SA fermentation in anaerobic flasks was unfettered by supplementary nutrients. However, fermentation in the benchtop fermenter devoid of supplementary nutrients resulted into 42% reduction in SA yield as well as lower SA productivities. Furthermore, a significant reduction of cell growth occurred in anerobic flasks at pH < 6.0, and complete termination of bacterial activity was noted at pH < 5.3. The highest SA titer, yield and productivity of 15.67g/L, 0.54g/g and 0.33g/L/h, respectively, was recorded from whey fermentation with MgCO3. The present study further highlights significant inhibitory effect of K2CO3 buffered medium on Actinobacillus succinogenes. Thus, we can claim that environmental pollution as well as costs of SA production from whey can be reduced by leveraging on whey residual nutrients to support the activity of Actinobacillus succinogenes.

Cytotoxicity, Antimicrobial Activity, Molecular Docking, Drug likeness and DFT Analysis of Benzo[c]phenanthridine Alkaloids from Roots of Zanthoxylum chalybeum

Zanthoxylum chalybeum (Rutaceae) is traditionally used to treat malaria, tuberculosis, intestinal problems and pneumonia. Roots extract was subjected to silica gel column chromatographic separation to afford four benzo[c]phenanthridines alkaloids (1-4), of which compounds (2) and (3) are reported herein for the first time from the species. Cytotoxicity analysis revealed chelerythrine (1) and dihydrochelerythrine (4) induced a significant reduction of cell growth of MDA-MB-231 and MCF-7 breast cancer cell lines in a dose-dependent manner. Chelerythrine (1) showed the highest potency against the aggressive and metastatic MDA-MB-231 cell line (IC50 = 3.616 ± 0.51 µM). The compounds showed the influence in the cell cycle in the MDA-MB-231 cell line by arresting some cells in the G2/M phase, preventing cells with damaged DNA from entering mitosis. Chelerythrine (1) showed promising antibacterial and antifungal activity against S. aureus and C. albicans with IC50 = 12.5 µg/mL and IC50 = 50 µg/mL, respectively. Molecular docking analysis of alkaloids (1-4) revealed lowest binding energy ranged from -6.5 to -7.5 and -6.1 to -6.4 Kcal/mol targeting E.coli DNA gyrase B and topoisomerase II α, respectively. The results obtained from molecular docking, drug-likeness properties, ADMET and DFT analysis agree with those obtained from experimental studies. Hence, chelerythrine (1) and dihydrochelerythrine (4) have proved to have promising activity against infectious diseases caused by microorganisms and human breast cancer cells, suggesting the potential use of the compounds as medicine which corroborate the traditional uses of the plant.

Discovery and Pharmacological Evaluation of STEAP4 as a Novel Target for HER2 Overexpressing Breast Cancer.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

Robust phenotypic maintenance of limb cells during heterogeneous culture in a physiologically relevant polymeric-based constructed graft system

A major challenge during the simultaneous regeneration of multiple tissues is the ability to maintain the phenotypic characteristics of distinct cell populations on one construct, especially in the presence of different exogenous soluble cues such as growth factors. Therefore, in this study, we questioned whether phenotypic maintenance over a distinct population of cells can be achieved by providing biomimetic structural cues relevant to each cell phenotype into the construct’s design and controlling the presentation of growth factors in a region-specific manner. To address this question, we developed a polymeric-based constructed graft system (CGS) as a physiologically relevant model that consists of three combined regions with distinct microstructures and growth factor types. Regions A and B of the CGS exhibited similar microstructures to the skin and soft tissues and contained rhPDGF-BB and rhIGF-I, while region C exhibited a similar microstructure to the bone tissue and contained rhBMP-2. Primary rat skin fibroblasts, soft tissue fibroblasts, and osteoblasts were then cultured on regions A, B, and C of the CGS, respectively and their phenotypic characteristics were evaluated in this heterogenous environment. In the absence of growth factors, we found that the structural cues presented in every region played a key role in maintaining the region-specific cell functions and heterogeneity during a heterogeneous culture. In the presence of growth factors, we found that spatially localizing the growth factors at their respective regions resulted in enhanced region-specific cell functions and maintained region-specific cell heterogeneity compared to supplementation, which resulted in a significant reduction of cell growth and loss of phenotype. Our data suggest that providing biomimetic structural cues relevant to each cell phenotype and controlling the presentation of growth factors play a crucial role in ensuring heterogeneity maintenance of distinct cell populations during a heterogeneous culture. The presented CGS herein provides a reliable platform for investigating different cells responses to heterogeneous culture in a physiologically relevant microenvironment. In addition, the model provides a unique platform for evaluating the feasibility and efficacy of different approaches for simultaneously delivering multiple growth factors or molecules from a single construct to achieve enhanced cell response while maintaining cellular heterogeneity during a heterogenous culture.

Supersaturated LFCS type III self-emulsifying delivery systems of sorafenib tosylate with improved biopharmaceutical performance: QbD-enabled development and evaluation.

The current studies investigate the application of quality by design-enabled type III self-emulsifying delivery system (Type III-SEDDS) of sorafenib tosylate (SFN) in improving its biopharmaceutical attributes. Initially, lipidic and emulsifying excipients were selected by carrying out solubility and phase titration experiments. After screening studies using Taguchi OA design, Type III-SEDDS were further optimised using D-optimal mixture design. The prepared formulations were assessed for globule size, zeta potential and percent of drug release. Following graphical optimisation, the optimum formulation was earmarked and further supersaturated to form saturated Type III-SEDDS (Sat-Type III-SEDDS) using a combination of HPMC and PVP to improve the stability of the formulation for a prolonged period. In vitro drug release of Type III-SEDDS study indicated approximately 8-fold improvement in dissolution rate over the pure powder drug. Cell uptake studies demonstrated higher uptake of dye-loaded Type III-SEDDS formulations in Caco-2 cells vis-à-vis plain dye. Cytotoxicity assay on Hep G2 cells revealed significant reduction in cell growth with Type III- and Sat-Type III-SEDDS vis-à-vis the pure drug. Furthermore, in situ permeation studies carried out using Wistar rats exhibited nearly 8.3- to 10.2-fold augmentation in permeation and absorption parameters of the drug from the Type III- and Sat-Type III-SEDDS, respectively, vis-à-vis the pure drug. Pharmacokinetic studies indicated nearly 3.98- and 3.62-fold improvement in AUC0-72, and 8.01- and 5.42-fold in Cmax, along with 0.25-fold decrease in Tmax of the drug from Type III- and Sat-Type III-SEDDS, respectively, in comparison with the SFN suspension. Furthermore, high degree of level A linear correlation was established between fractions of drug dissolved (in vitro) and of drug absorbed (in vivo) at the corresponding time points for Sat-Type III-SEDDS and pure drug, whereas the Type III-SEDDS exhibited a nonlinear relationship. Stability studies indicated the robustness of Sat-Type III-SEDDS, when stored at 25°C for 3months. Overall, the manuscript documents the successful systematic development of SFN-loaded Sat-Type III-SEDDS with distinctly improved biopharmaceutical performance. Graphical abstract.

Abstract 1872: Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells

Abstract Oleuropein is one of the two main phenolic compounds derived from olive leaves. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, and increase apoptosis in many types of cancer cells, suggesting its possible efficacy as an anti-cancer treatments. The purpose of our study was to examine the effects of oleuropein extract on cell growth and viability of the K562 human leukemia cell line, and to investigate possible molecular mechanisms of action. Oleuropein of 98% purity was used for our studies. We first demonstrated that K562 cells treated with 200 and 400 μg/ml oleuropein exhibited a significant reduction in cell growth and viability after four days as compared to ethanol-treated controls. Cells treated with 200μg/ml oleuropein showed decreased cell proliferation and viability after two days, and exhibited approximately 90% reduction in cell density and viability after four days of treatment. A concentration of 400 μg/ml oleuropein resulted in 100% toxicity by day four. We also examined the effects of oleuropein on cellular cytotoxicity using an LDH release assay and found a two-fold elevation in LDH release within 24 hours of treatment with 200 μg/mL oleuropein. To begin to explore a mechanisms of action, we used western blotting to examine known antioxidant and signaling pathways for possible regulation by oleuropein in these cells. We examined expression of the Peroxiredoxin (Prdx) family of proteins, a group of thiol-specific antioxidants found to be elevated in many types of cancer. We found that 200 ug/ml oleuropein reduced Prdx1 expression by about 50% after eight and twenty-four hours, while having no significant effect on expression levels of Prdx-2, -3, -4, and -6. We are currently investigating the mechanism of cell death induced by oleuropein, as well as the role of Prdx1 in oleuropein-induced toxicity. Together with previously reported cancer-cell specific effects, this research provides a promising new avenue for the use of natural products as anti-cancer agents. Citation Format: Natalie Fulco, Sophia Fagan, Shelley A. Phelan. Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1872.

Extracts from Cell Suspension Cultures of Strawberry (Fragaria x ananassa Duch): Cytotoxic Effects on Human Cancer Cells.

Natural compounds are emerging as agents for the treatment of malignant diseases. We previously showed that extracts from in vitro cell suspension cultures of strawberry reduced murine melanoma cell proliferation, as shown for fruit extracts. In this work, chromatographic, mass spectrometric, and spectrophotometric analyses were carried out to identify the bioactive compound exerting the detected cytotoxic activity. Moreover, aiming to confirm the anti-proliferative activity of the extracts against both paediatric and adult human tumors, cytotoxic experiments were performed on neuroblastoma, colon, and cervix carcinoma cell lines. Extracts from in vitro cell suspension cultures of strawberry induced a statistically significant reduction of cell growth in all the tumor cell lines tested. Interestingly, human fibroblasts from healthy donors were not subjected to this cytotoxic effect, highlighting the importance of further preclinical investigations. The accurate mass measurement, fragmentation patterns, and characteristic mass spectra and mass losses, together with the differences in chromatographic retention times and absorbance spectra, led us to hypothesize that the compound acting as an anti-proliferative agent could be a novel acetal dihydrofurofuran derivative (C8H10O3, molecular mass 154.0630 amu)

Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth.

Hepatocellular carcinoma (HCC) is a common and deadly cancer with limited treatment options. Through genome-wide growth depletion screens using clustered regularly interspaced short palindromic repeats and expression profiling of primary HCC tumors, we identified 13 clinically relevant target genes with therapeutic potential. Subsequent functional annotation analysis revealed significant enrichment of these 13 genes in the cell cycle, cell death, and survival pathways. Non–structural maintenance of chromosomes condensin I complex subunit G (NCAPG) was ranked the highest among the depletion screens and multiple HCC expression datasets. Transient inhibition of NCAPG using specific small interfering RNAs resulted in a significant reduction in cell growth, migration, and the down-regulation of mitochondrial gene expression in vitro. Small homologous RNA–mediated knockdown of NCAPG significantly impaired cell viability, caused aberrant mitotic division, fragmented the mitochondrial network, and increased cell death in vitro. HCC cells with a reduced expression of NCAPG formed significantly smaller xenograft tumors in vivo. Importantly, high NCAPG expression was significantly associated with poorer overall and disease-free survival in HCC patients. High NCAPG expression is a novel prognostic biomarker to predict HCC early recurrence after surgical resection. In conclusion, NCAPG is an essential gene for HCC tumor cell survival. It represents a promising novel target for treating HCC and a prognostic biomarker for clinical management of HCC.—Wang, Y., Gao, B., Tan, P. Y., Handoko, Y. A., Sekar, K., Deivasigamani, A., Seshachalam, V. P., OuYang, H.-Y., Shi, M., Xie, C., Goh, B. K. P., Ooi, L. L., Hui, K. M. Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth.

Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells

Oleuropein is one of the two main phenolic compounds derived from olive leaves. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, and increase apoptosis in many types of cancer cells, suggesting its possible efficacy as an anti‐cancer treatments. The purpose of our study was to examine the effects of oleuropein extract on cell growth and viability of the K562 human leukemia cell line, and to investigate possible molecular mechanisms of action. Oleuropein of 98% purity was used for our studies. We first demonstrated that K562 cells treated with 200 and 400 μg/ml oleuropein exhibited a significant reduction in cell growth and viability after four days as compared to ethanol‐treated controls. Cells treated with 200μg/ml oleuropein showed decreased cell proliferation and viability after two days, and exhibited approximately 90% reduction in cell density and viability after four days of treatment. A concentration of 400 μg/ml oleuropein resulted in 100% toxicity by day four. We also examined the effects of oleuropein on cellular cytotoxicity using an LDH release assay and found a two‐fold elevation in LDH release within 24 hours of treatment with 200 μg/mL oleuropein. To begin to explore a mechanisms of action, we used western blotting to examine known antioxidant and signaling pathways for possible regulation by oleuropein in these cells. We examined expression of the Peroxiredoxin (Prdx) family of proteins, a group of thiol‐specific antioxidants found to be elevated in many types of cancer. We found that 200 ug/ml oleuropein reduced Prdx1 expression by about 50% after eight and twenty‐four hours, while having no significant effect on expression levels of Prdx‐2, ‐3, ‐4, and ‐6. We are currently investigating the mechanism of cell death induced by oleuropein, as well as the role of Prdx1 in oleuropein‐induced toxicity. Together with previously reported cancer‐cell specific effects, this research provides a promising new avenue for the use of natural products as anti‐cancer agents.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FOXM1 and the NPM-ALK/STAT3 Axis Form a Novel Positive Feedback Loop in Promoting the Oncogenesis of ALK-Positive Anaplastic Large Cell Lymphoma

Peng Wang1, Moinul Haque2, Jing Li2,3, Yung-Hsing Huang2, Meaad Almowaled2, Carter Bargar4, Adam Karpf4, Will Chen2, Suzanne Turner5 and Raymond Lai2,61Division of Hematology, Dept of Medicine, University of Alberta, Edmonton, 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton Alberta, Canada; 3Electron Microscopy Center, Basic Medical Science College, Harbin Medical University, Harbin, Heilongjiang, China; 4Eppley Institute and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, USA; 5Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK;6Department of Oncology, University of Alberta, Edmonton, Alberta, Canada1. Backgrounds and AimsForkhead Box M1 (FOXM1) is a transcription factor implicated in the pathogenesis of solid tumors, and it has been shown to promote cell-cycle progression, stem cell renewal and chemotherapeutic resistance in cancer cells. Nonetheless, the biological significance of FOXM1 in hematologic malignancies has not been extensively studied. Here, we studied the expression and role of FOXM1 in ALK-positive anaplastic large cell lymphoma (ALK+ALCL).2 Methods and ResultsIn contrast with normal lymphocytes, FOXM1 was highly expressed in all ALK+ALCL cell lines (5/5), tumors from patients (6/6) and tumors arising from NPM-ALK transgenic mice. Experiments using nuclear/cytoplasmic fractionation, immunocytochemistry and reporter assays had provided evidence that FOXM1 is transcriptionally active in ALK+ALCL. Down-regulation of FOXM1 expression using shRNA and a pharmacologic agent (thiostrepton) resulted in a significant reduction in cell growth, colony formation in soft agar and cell-cycle arrest in ALK+ALCL cells. Further studies revealed that the oncogenic potential of FOXM1 is linked to substantial increases in the phosphorylation/activation status of NPM-ALK and STAT3, and the upregulations of a host of cytokines that have been previously shown to activate the NPM-ALK/STAT3 axis, including IGF-1, IL9 and IL21. Using co-immunoprecipitation, we found that NPM-ALK binds to FOXM1 in the nucleus of ALK+ALCL cells. Importantly, the binding of NPM-ALK to FOXM1 promotes the DNA binding ability and transcriptional activity of FOXM1, and functional inhibition of NPM-ALK using crizotinib or depletion of NPM-ALK using siRNA in ALK+ALCL cells significantly decreased the transcriptional activity of FOXM1.Conclusions:In conclusion, we have identified a novel oncogenic feedback loop involving FOXM1 and the NPM-ALK/STAT3 axis in ALK+ALCL. This study has revealed the first clear example in which NPM-ALK exerts important oncogenic functions in the nuclei of ALK+ALCL cells, by means of its binding to an oncogenic transcription factor so as to promote its DNA binding and transcription activity. DisclosuresNo relevant conflicts of interest to declare.

ALPK1 Expression Is Associated with Lymph Node Metastasis and Tumor Growth in Oral Squamous Cell Carcinoma Patients

Oral squamous cell carcinoma (OSCC) is the most common malignant cancer, with high mortality rates in advanced stages. Recent studies have shown that the expression of ALPK1 mRNA and its inhibitory differentiation function are associated with cancer progression. However, the expression and clinicopathologic features of ALPK1 in OSCC remain unexplored. Herein, the authors investigated the expression patterns of ALPK1 in 39 matched OSCC patients and examined the relationship between ALPK1 protein expression and clinicopathologic factors using immunohistochemical scores. Using Western blot analysis, ALPK1 expression was found to be significantly higher in tumor tissues than that in nontumor tissues. Through an immunoreactive scoring system, a significantly higher number of advanced-stage tumor size T4 and lymph node metastasis N2 exhibited higher ALPK1 expression levels than that exhibited by T1/T2/T3 tumors and N0/N1. In addition, ALPK1 protein expression was aberrant in malignant oral cancer cell lines compared with that in pre-malignant oral epithelial cells, whereas minimal expression was observed in normal oral epithelial cells. Knockdown of ALPK1 resulted in a significant reduction in cell growth, migration, and invasion capacity invitro. Consequently, expression of N-cadherin and vimentin decreased in ALPK1-deficient cells. Thus, these results suggest that ALPK1 serves as a potential biomarker and target for OSCC development in late stages.

Abstract 4388: Anti-cancer effects of oleuropein olive leaf extract in K562 leukemia cells

Abstract Olive leaves come from a Mediterranean plant that many believe to have positive effects on human health, including anti-cancer properties. Oleuropein is one of the two main phenolic compounds derived from olive leaves, and it has been shown to increase apoptosis in many types of cancer cells. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, suggesting that the compound could be effective in anti-cancer treatments. The purpose of this study was to examine the effects of oleuropein extract on cell growth and viability in K562 human leukemia cells, and begin to investigate possible molecular mechanisms of action. We purified our own extract from olive leaves for these studies. Oleuropein was microwave extracted from olive leaf powder using methanol and water as solvent. The oleuropein was successfully identified using HPLC to characterize elution time in comparison to an authentic standard. HPLC was then used in purification and fractions were lyophilized. Isolated oleuropein was greater than 94% in comparison to the authentic standard. We next demonstrated that K562 cells treated with 50-150 ug/ml of oleuropein exhibited significant reduction in cell growth and cell viability after four days when compared to 70% ethanol-treated controls. This effect was markedly greater after 6 days . Current studies are being conducted to determine the most effective concentration and duration of treatment for oleuropein-induced cell death. We are also examining cellular cyytotoxicity and cell cycle progression in these experiments using LDH assay and cell cycle staging, and further studies are being conducted to examine inflammatory and antioxidant pathways for their involvement in the anti-cancer effect of the compound in these cells. Given the observation that many of the cytotoxic effects of oleuropein are cancer-cell specific, this research provides a promising new avenue for the use of natural products as anti-cancer agents. Citation Format: Sophia Fagan, Natalie Fulco, Justin Gilbertson, Aaron R. Van Dyke, Shelley A. Phelan. Anti-cancer effects of oleuropein olive leaf extract in K562 leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4388.

Abstract 4629: Synergistic growth inhibitor effect on a patient derived NF1 pilocytic astrocytoma cell line with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor trametinib

Abstract Pediatric low grade glioma (PLGG) is the most common brain tumor of childhood. If the tumor is located in a region of the brain that is not accessible for surgical resection, additional therapies are needed. We and others have identified mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We hypothesized that TAK228 and trametinib would show synergistic effects in vitro and in vivo PLGG models. We treated in vitro four different patient-derived PLGG cell lines with TAK228, trametinib, or vehicle control: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAFfusion), Res186 and Res259 (both cell lines with mTOR and MAPK activation). In vivo, BT40 (BRAFV600E) tumor cells were investigated with both agents in immunodeficient mice. Cell growth was investigated using MTS-assay compared to vehicle control. Activation of MAPK pathway was detected via Western Blot by phosphorylated ERK compared to total ERK, and β-actin. mTOR pathway was investigated with pAKT, pS6, and p4E-BP1 compared to the total protein amount and β-actin. In all of the cell lines, treatment with TAK228 or trametinib reduces cell growth and proliferation in a dose and time depended manner. We have found a robust synergistic (via Chou-Talalay method) effect for JHH_NF1_PA1, Res186, and Res259 cells in clinically relevant doses of both drugs (5nM, 10nM, 20nM). BT66_SV40/hTERT cells have a significant reduction in cell growth under TAK228 treatment after 4 days by up to 70% (p&amp;lt;0.001; via one-way ANOVA), but not under trametinib treatment. Interestingly for this cell line, the MAPK pathway was inactivated in all tested trametinib doses (≥1nM) and in combination treatment. In all cell lines trametinib treatment leads to a pERK inactivation at low nM levels. TAK228 leads to an inactivation of mTORC1 and mTORC2 in all four tested cells lines. In TAK228 treated cells, there was compensatory activation of pERK, which was reduced when trametinib was added. Apoptosis induction was verified through cleaved PARP via western blot and CC-3 via immunocytochemistry. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p&amp;lt;0.001). After determination the optimal dosing schedule for TAK228 (1mg/kg/every other day), trametinib (3mg/kg/daily) and combination, BT40 transplanted nude mice are investigated for tumor size and survival. Our results show that PLGG-derived cell lines are sensitive to TAK228 and trametinib treatment. The increased MAP kinase activity we identified after TAK228 treatment, suggests a compensatory mechanism that may render these cells especially sensitive to treatment with both TORC1/2 and MEK inhibitors. The ongoing in vivo experimentation will provide a pre-clinical rationale for combination therapy of these agents in PLGG. Citation Format: Antje Arnold, Ming Yuan, Fausto J. Rodriguez, Charles G. Eberhart, Eric H. Raabe. Synergistic growth inhibitor effect on a patient derived NF1 pilocytic astrocytoma cell line with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor trametinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4629.