bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying novel molecular vulnerabilities to PTK2/FAK inhibition in G α q‐driven uveal melanoma using a kinome‐wide CRISPR/Cas9 screen
Authors: Kishore Ayush , Feng Xiaodong , Arang Nadia , Wu Xingyu , Pachter Jonathan , Schlaepfer David D. , Tamayo Pablo , Cheng Qianming , Gutkind J. Silvio
Published: June 2021

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. It is diagnosed in about 2,500 adults in the United States every year. Approximately 50% of UM patients develop liver metastasis within 5–10 years after diagnosis, independently of successful treatment of the primary lesions. Activating mutations in GNAQ and GNA11 (encoding GTPase deficient/constitutively active Gαq proteins) were identified in ~90% of UM; where they act as driver oncogenes. MEK inhibition with selumetinib or trametinib has nearly no impact on the overall survival of UM patients. Therefore, there is an urgent need to understand mechanistically how Gαq promotes UM progression in order to develop new therapeutics. In a recent study, we have performed an integrated bioinformatics analysis of gene interaction networks and identified PTK2, encoding the tyrosine kinase: Focal Adhesion Kinase (FAK) as a candidate gene highly predicted to contribute to Gαq‐driven UM progression. Using a variety of biochemical and cell‐based assays, we established that FAK is indeed a pivotal signaling node downstream of oncogenic Gαq that mediates UM proliferation through the activity of the growth‐promoting transcription factor Yap. Notably, our studies with UM xenograft mouse models demonstrated that FAK blockade by VS‐4718, a new generation orally‐bioavailable FAK inhibitor, dramatically reduced tumor volume. The major objective of the current study is to further delineate the Gαq‐FAK signaling axis with interest in identifying novel synthetic lethal interactions to FAK inhibition in UM. To this end, we conducted a kinome‐wide CRISPR/Cas9 screen using VS‐4718 in UM cell line OMM1.5 to pinpoint molecular vulnerabilities in the Gαq‐FAK onco‐circuitry. Positive hits from our gene‐drug screen will be presented, which shed light on the Gαq‐FAK network and may serve as potential co‐targets to FAK inhibition in UM clinical therapy.

Support or Funding Information

Cancer Biology, Informatics & Omics Training Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
2D vs 3D – Triple negative breast cancer spheroid formation induces quantitative heterogeneity of VEGF and PDGF receptor profiles and modulates cytosolic phosphorylation
Authors: Oyirifi Ashley , Joyce Kaitlyn M. , Helferich William G. , Imoukhuede Princess I
Published: June 2021

Micro‐tumor spheres (MTS) have been considered a phenotypic intermediate between 2‐dimensional cell culture and tumor explants from living organisms. However, limited information exists for understanding membrane bound antigens of live cells in MTS. These antigens include several families of transmembrane proteins fundamental to signal transduction, cell communication, and adhesion processes and are thereby identified as key therapeutic targets in malignant cells. This study couples MTS cell culture and a high throughput method for live single cell receptoromic analysis, to compare MTS and monolayer cell surface densities of the PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 tumor angiogenesis regulating receptors on MDB‐MB‐231.

Cumulatively, the average density of PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 in MTS is 1.7‐fold higher in tumor only MTS when compared to monolayer cells. When cells are analyzed on a cell‐by‐cell basis, 2‐component Gaussian mixture models predict means of high‐density and low‐density MTS sub‐populations within PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 frequency distributions. The high density to low density means show 3.9‐, 1.6‐, 1.1‐, and 3.2‐fold differences for PDGFRα, PDGFRβ, VEGFR1, and VEGFR2, respectively. Finally, an analysis of MTS intracellular phosphorylation profiles preliminarily reveals attenuated phosphorylation, including reduced activation of the MAPK pathway, in MDA‐MB‐231 MTS simulated by a 20pg/mL VEGFA solution. Together, the current data suggests that MTS may provide a biologically relevant representation of breast cancer cells' VEGFR and PDGFR signaling axes through altering cell surface receptor density and heterogeneity. Our continued work will investigate the effects of varying VEGA concentrations on MTS signal transduction.

Support or Funding Information

This research was supported by NIEHS grant (T32 ES007326).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decreased Expression of Enolase‐I Promotes Cisplatin Resistance of Ovarian Cancer Cells
Authors: Rabelo‐Fernández Robert Joe , Vivas‐Mejia Pablo , Santana‐Rivera Yasmarie
Published: June 2021

Platinum‐containing anticancer drugs has been used for more than four decades has effective treatment for ovarian cancer and many others solid tumors. However, the initial positive response to therapy is limited by development of resistance to cisplatin treatment. Although many mechanism of cisplatin resistance have been proposed, the molecular mechanism leading to the cisplatin resistance in ovarian cancer tumors has not fully understood. In this study, we sought to identify novel proteins associated with the cisplatin resistance of ovarian cancer cells. Quantitative proteomic analysis revealed low protein levels of Enolase‐1 (ENO1) in cisplatin resistant (A2780CP20) ovarian cancer cells compared to its counterpart cisplatin sensitive (A2780) cells. Small‐interference RNA (siRNA)‐targeted ENO1 showed a reduction in the sensitivity of ovarian cancer cells to cisplatin treatment. Contrasting, ectopic expression of ENO1 in cisplatin resistant ovarian cancer cells reduced the sensitivity of these cells to cisplatin treatment. Intracellular glucose uptake were measurements in pairs of cisplatin sensitive and cisplatin resistant cells. The glucose content levels were higher in all cisplatin resistant cells compared with their sensitive counterparts. Overall, these results suggest that low levels of ENO1 are associated with the cisplatin resistance of ovarian cancer cells. Therefore, targeting the glycolytic pathways could be a strategy to overcome the cisplatin resistance of women with advanced and drug resistant ovarian cancer.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pimavanserin tartrate: A potential drug for pancreatic cancer therapy in future
Authors: Ramachandran Sharavan , Srivastava Sanjay K
Published: June 2021

Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. In the current study, we evaluated the anti‐cancer effects of pimavanserin tartrate (PVT), a drug used for the treatment of Parkinson's disease psychosis. Our observations indicated that, PVT significantly suppressed the proliferation of pancreatic cancer cells by inducing apoptosis without exerting any cytotoxic effects in normal human pancreatic ductal epithelial (HPDE‐6) cells. The colony forming ability of pancreatic cancer cells was significantly inhibited with PVT treatment. Anti‐proliferative and apoptosis inducing effects of PVT were mediated by the inhibition of pAkt (Ser473), Akt, Gli1, Oct‐4, SOX‐2, NANOG and c‐Myc. Akt and Oct‐4 inhibition by PVT treatment was further validated by immunofluorescence analysis. Apoptotic effects of PVT was confirmed by increase in cleavage of caspase‐3 and PARP. Moreover, PVT inhibited the formation of tumorspheres in PANC1 pancreatic cancer cells. Pharmacologically inhibiting or genetically knocking out Akt or Gli1 enhanced the growth suppressive effects of PVT in pancreatic cancer cells. Subsequently, we evaluated the effects of PVT in gemcitabine‐resistant cells. Our results demonstrated that, PVT reduced the survival of MIAPaCa2 gemcitabine‐resistant cells in a concentration and time‐dependent manner. Further mechanistic analysis indicated that, PVT suppressed the phosphorylation of Akt at Ser 473 and inhibited the expression of Gli1, Oct‐4 and c‐Myc in MIAPaCa2 gemcitabine‐resistant cells. Moreover, PVT increased the cleavage of caspase‐3 and PARP as an indicator of apoptosis. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 75%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Taken together, our results indicate that pancreatic tumor growth suppression by PVT is orchestrated by inhibition of Akt/Gli1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer.

Support or Funding Information

Supported in part by R01 grant CA129038, awarded to (S.K.S.) by the National Cancer Institute, Syngenta fellowship grant awarded by Syngenta pharmaceuticals.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial RhoA Regulates Breast Cancer Metastasis
Authors: Sajib Md Sanaullah , Zahra Fatema Tuz , Park Jee Hyun , Tullar Paul , Srivastava Sanjay K. , Bickel Ulrich , Mikelis Constantinos M
Published: June 2021

Metastasis is the cause of more than 90% of cancer‐related deaths. An important step in metastatic process is the migration of the disseminating cancer cells through the endothelial monolayer during entrance or exit from the vasculature. Activation of endothelial RhoA‐ROCK pathway has been implicated in the regulation of vascular permeability in various pathophysiological conditions. In the present study, we have investigated the role of this pathway in trans‐endothelial migration and metastasis of breast cancer cells. RhoA activation was identified by RhoA pull‐down experiments of murine and human breast cancer cell supernatant on primary endothelial cells. Cancer cell trans‐endothelial migration efficiency was explored through an in vitro transwell‐based two‐cell co‐culture model. RhoA signaling pathway was blocked by both pharmacological inhibition and knockdown experiments. In vivo metastatic potential was identified by experimental metastasis models of murine and human breast cancer cell lines using endothelial‐specific RhoA‐deficient mice or clinically relevant ROCK inhibitors. We identified that the diverse metastatic potential of a panel of human breast cancer cell lines correlated with the RhoA activation efficiency of their supernatant on primary endothelial cells. Secretome analysis revealed that IL‐8 (over)expression was relevant to breast cancer cell aggressiveness and its functionality was tested with gain‐and loss‐of‐function experiments. In vivo, experimental metastasis experiments with the syngeneic breast cancer cell line E0771 showed that endothelial RhoA deficiency decreased the metastatic potential. Similar inhibition was obtained with treatment with the clinically‐relevant ROCK inhibitor, Fasudil, in experimental metastasis experiments of murine and human cell lines using immune‐competent and immune‐deficient mice respectively. Collectively, our in vitro and in vivo findings demonstrate that the endothelial RhoA‐ROCK pathway affects breast cancer trans‐endothelial migration and metastasis and propose endothelial RhoA inhibition as a novel therapy for breast cancer metastases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Tyrosine Phosphatase Receptor Type F Promotes Wnt Signaling in Colorectal Cancer
Authors: Stevens Ashley , Gan Tong , Gao Tianyan
Published: June 2021

Colorectal cancer is the second leading cause of cancer deaths in the United States. A major mechanism behind the formation of colon cancer is the activation of Wnt signaling through a mutation in the APC or β‐catenin gene, which leads to accumulation of active β‐catenin. Aberrant signaling of the Wnt pathway has been linked to the initiation and progression of colorectal cancer. A major mechanism of control for the Wnt signaling pathway is protein phosphorylation. A fine balance between phosphorylation and dephosphorylation exist to keep the cell balanced depending on its needs. Protein phosphatases are important in regulating signaling pathways by dephosphorylating a large number of cellular substrates. In this study, we investigated the functional importance of PTPRF, a member of protein tyrosine phosphatase receptor type 2A, in regulating Wnt signaling in colon cancer. Bioinformatics analysis indicated that PTPRF is highly expressed in the intestinal epithelium. Silencing PTPRF in colon cancer cells resulted in a decrease in the expression of Wnt target genes. As a consequence, the rate of cell proliferation was significantly decreased in PTPRF knockdown cells. To determine the molecular mechanism, we examined the internalization process of Wnt‐Fzd‐LRP6 receptor complex, a key mode of regulation for the Wnt pathway. Specifically, we monitored the time course of LRP6‐GFP tracking in control and PTPRF knockdown cells upon Wnt stimulation. Our preliminary confocal microscopy results showed that the amount of LRP6‐GFP protein detected at the cell surface was decreased basally and the time course of LRP6‐GFP endocytosis was altered upon Wnt treatment in PTPRF knockdown cells. Interestingly, we found that PTPRF was co‐localized with LRP6‐GFP at both endocytic vesicles and plasma membrane in colon cancer cells, and Wnt treatment promoted internalization of PTPRF. Taken together, our study has identified PTPRF as a novel regulator of Wnt signaling by controlling the endocytosis of LRP6 receptor in colorectal cancer.

Support or Funding Information

This work was supported by R01CA133429 and R01CA208343.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of Inhibitors of NHE1 Activation as Potential Adjuvant Therapies in the Treatment of Ovarian Cancer
Authors: Corradi Anna R. , Wallert Mark A.
Published: June 2021

Each year in the United States over 22,000 women are diagnosed with ovarian cancer leading to more than 14,000 deaths. Increases in the expression and activity Na+‐H+ Exchanger Isoform 1 (NHE1) was recently demonstrated to regulate cell proliferation in ovarian cancer patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. NHE1 is hyperactivated in an array of cancer types leading to a pH inversion that supports both cell proliferation and invasion. One mechanism for this NHE1 hyperactivation is phosphorylation of the regulatory domain by a range of protein kinases. A significant issue in ovarian cancer therapy is the development of drug resistance and disease recurrence. In an effort to combat these outcomes, it is hypothesized that combining common ovarian cancer therapeutic agents along with compounds that indirectly inhibit NHE1 activation will enhance the ability of the therapeutic agents to slow ovarian cancer progression. Two ovarian adenocarcinoma cell lines; CAOV‐3 and SKOV‐3 are used in this research. Using an XTT proliferation assay, the impact of different primary and second‐line ovarian cancer therapeutics alone and in combination with specific kinase inhibitors was evaluated. The therapeutics evaluated are: 1) Rucaparib, 2) Olaparib, 3) Paclitaxel, 4) Cisplatin, and 5) Carboplatin. An initial study of the protein kinases that regulate NHE1 activity was previously conducted. Four protein kinases that phosphorylate NHE1 at five specific locations: ROCK (RhoA‐Associated Kinase) at T653, RSK (Ribosomal S6 Kinase) at S703, AKT (Protein Kinase B) at S648, and ERK (Extracellular‐Signal Regulated Kinase) at S770/S771 were evaluated. The kinase inhibitors used were: MK220 (AKT), SCH772984 (ERK), Y2732 (ROCK), and BID1870 (RSK). Each of the pharmaceutical agents and the kinase inhibitors decreased cell proliferation independently in each of the cell lines. Proliferation data will be presented for the therapeutics independently and in the presence of kinase inhibitors. This work has also been expanded to a three‐dimensional environment, investigating the impact of these combination therapies in a spheroid assay. These data indicate that inhibition of NHE1 activating kinases may serve as a potential therapeutic target in Ovarian Cancer treatment.

Support or Funding Information

Lueken Family Cancer Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ABC Transporter‐Mediated Multidrug Resistance in Prostate Cancer Cells
Authors: Famuyiwa Toluleke O
Published: June 2021

Background

Prostate cancer is the second most diagnosed cancer in men. The high incidence of prostate cancer has been attributed to a failure in chemotherapy. Studies show that energized ATP Binding Cassette proteins cause 50% of the failure in chemotherapy. This study focuses on inhibiting ATP Binding Cassette (ABC) protein‐mediated drug resistance in prostate cancer treatment by using Poly Lactic‐co‐Glycolic Acid (PLGA) nanoparticle to deliver SC‐514 and 3‐Bromopyruvate (3‐BPA).

Objective of study

This study aims to; (i) investigate the potential interaction between 3‐BPA and SC‐514, (ii) reduce treatment induced ABC mediated multidrug resistance (MDR), and (iii) investigate the signaling pathways involved in ABC transporter‐mediated MDR.

Method

Bioassays used in this preliminary study include trypan blue exclusion, MTT tetrazolium, NBT, cell titer glow, fluorescence microscopy, multidrug resistance efflux.

Results

One‐way ANOVA comparing the impact of 3‐BPA, SC‐514 and the combination of 3‐BPA and SC‐514 on LNCaP cells at 24hrs of treatment shows that p=0.00023. Time‐dependent treatment of LNCaP cells with 3‐BPA, SC‐514, and a combination of 3‐BPA and SC‐514 after regression analysis shows the following p values: 24hrs (0.00023), 48hrs (0.00003), 72hrs (0.000000152), 96hrs (0.000000049). ROS level of LNCaP cells treated with 3‐BPA (r=−0.5, p=0.11), SC‐514 (, r=− 0.72 p=0.04,) and 3‐BPA + SC‐514 (r=−0.58, p=0.04) shows no significant difference in ROS modulation (p=0.54). There was a weak to moderate correlation between ROS released and cell death. There was a weak correlation between percentage ROS induced and percentage apoptotic death. There was a positive correlation between the concentration of the drug and cell death. 3‐BPA and/or SC‐514 depleted intracellular ATP in DU‐145 cells. There was a time‐dependent effect of 3‐BPA and/or SC‐514 on cell death. SC‐514 and/or 3‐BPA are substrates for MDR1. 3‐BPA and/or SC‐514 potentially block MDR1. DU‐145 prostate cancer cells efflux dyes within 15 minutes.

Conclusion

3‐BPA and SC‐514 has the potential to inhibit multidrug resistance by reducing the intracellular ATP available to ATP binding cassette proteins. Apoptotic induction in DU‐145 prostate cancer cells exists via a mechanism other than reactive oxygen species (ROS) induction.

Support or Funding Information

Fellowships and grants

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Antimicrobial Properties of Essential and Natural Oils on Cronobacter Species
Authors: Whitehead Nickolas T. , Kanipes Margaret I. , Fakayode Sayo O.
Published: June 2021

The use of essential and natural oils are becoming increasingly popular due to their antimicrobial, therapeutic antioxidant properties. Research has shown that essential and natural oils are effective against a variety of bacteria. However, not much experimentation has been conducted to investigate the antibacterial properties of essential oils and natural oils on Cronobacter species. This study evaluates the antibacterial property of ten essential and natural oil samples on Cronobacter species using a modified disk diffusion assay using a ciprofloxacin as control. The results of the study shows differences in the antibacterial properties of essential and natural oils. The essential oils that inhibited cell growth of Cronobacter muytjensii were lemon eucalyptus oil (Eucalyptus citriadora), lemon oil (Citrus limonum), tea tree lemon oil (Leptospermum petersonii), and wintergreen oil (Gaultheria fragrantissima). Neem oil (Azadirachta indica) (a natural oil), displayed signs of reduced growth in the bacteria. The inhibitory properties of the aforementioned oils will be discussed.

Support or Funding Information

North Carolina A&T University Research Initiative for Scientific Enhancement (RiSE) Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypoglycemia effect of drink containing Guava Leaf Extract in Rats with type 2 diabetic rats
Authors: Huang Hui‐Yu , Chen Li‐Han , Chan Ching‐Hung
Published: June 2021

This study investigated the effect of drink containing Guava Leaf Extract (GLE) (Psidium guajava Linn.) on hypoglycemia and glucose metabolism in type 2 diabetic rats. High fat‐fructose Diet and Low‐dose streptozotocin (STZ) and nicotinamide were given and injected into Sprague‐Dawley (SD) rats to induce type 2 diabetes. Fifty rats were equally assigned into diabetes mellitus group (DM), positive control group (PC), low‐dose GLE group (L), medium‐dose GLE group (M), and high‐dose GLE group (H) respectively. We found that GLE progressively decreased fasting blood glucose and OGTT (□ < 0.01). Elevated pro‐inflammatory cytokines, interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNF‐α), were controlled by L, M and H group. Antioxidant activity was increased in L, M and H group. Experimental evidences demonstrate that GLE may be effective alternative medicine to prevent HFFD‐induced hyperglycemia and hyperlipidemia, possibly associated with anti‐inflammatory and antioxidant efficacies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating a Novel Metabolic Pathway for Bacteria to Utilize L‐Ascorbate as its Carbon Source
Authors: Kim Peter , Stack Tyler , Carter Michael
Published: June 2021

L‐Ascorbate (Vitamin C) is an abundant yet under‐researched carbon source present in the environment. L‐Ascorbate is a well‐known antioxidant that has significant effects of promoting tissue repair, strengthening bones, and boosting the immune system in mammals. Ralstonia eutropha H16 (Cupriavidus necator) is a saprotrophic soil bacterium that is commonly known for its ability to metabolize a great variety and number of substrates for growth and production of polyhydroxybutyrate (bioplastics). Ralstonia eutropha (ReH16) growth with L‐ascorbate was observed, but the pathway by which ReH16 metabolizes L‐ascorbate is unknown. Transcripts were quantified using RNAseq from cells grown with L‐ascorbate or other carbon sources. Putative pathway genes were identified as genes with transcripts upregulated greater than 50‐fold during L‐ascorbate growth. Transformation, conjugation, homologous recombination, and PCR analysis were used to generate and confirm targeted deletions of two pathway genes [h16_B0212 and h16_B1217]. Mutants of both genes were unable to grow with L‐ascorbate as their sole carbon source. Collaborators have characterized both enzymes. H16_B0212 is an aldehyde dehydrogenase that catalyzes oxidation of the δ‐carbon of α‐ketoglutarate semialdehyde. H16_B1217 is an enzyme that catalyzes the formation of a β‐lactone ring and transfer of a carboxyl group. Characterizing the L‐ascorbate metabolic pathway in Ralsontia eutropha will provide insight into a third, yet unknown pathway for L‐ascorbate metabolism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tempol, a superoxide dismutase mimetic agent, improves dystrophic phenotype in the diaphragm muscle of mdx mice
Authors: Minatel Elaine , de Almeida Hermes Túlio , Mâncio Rafael Dias , Macedo Aline Barbosa , Mizobuti Daniela Sayuri , da Rocha Guilherme Luiz
Published: June 2021

Duchenne muscular dystrophy (DMD) is a progressive and lethal X‐linked myopathy, in which the oxidative stress and abnormal reactive oxygen species (ROS) production plays a key role in its pathophysiology. Despite intense efforts, no cure is currently available for DMD and glucocorticoids are the only effective drug to slow down disease progression. Thus, in the present study we investigated Tempol (a superoxide dismutase mimetic agent) effects on the diaphragm muscle of mdx mice, an experimental model of DMD. Mdx mice were randomly divided into two groups: mdxS, the control group receiving intraperitoneal (i.p.) injections of saline solution (100μL) and mdxT, the treated group receiving i.p. injections of Tempol (100 mg/kg). C57BL/10 mice were also used as controls. The results showed that Tempol treatment prevented muscle damage indicated by gain in muscle strength, decrease in the IgG uptake by muscle fibers and reduction of regenerated muscle fibers indicated by the central nuclei. It also had an effect on the inflammatory process, reducing the inflammatory area, macrophage staining and decreasing TNF‐α, IL‐1β and IL‐6 levels. Regarding oxidative stress, Tempol treatment was effective in reducing ROS and lipid peroxidation in the dystrophic diaphragm muscle. The findings presented here show that Tempol treatment improves dystrophic phenotype, supporting its use as a potential therapeutic strategy in DMD.

Support or Funding Information

FAPESP #2017/01638‐0CAPES FAEPEX

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alda‐1 shields mitochondrial dynamic proteins from hyperoxia via Aldh2 activation
Authors: Patil Sahebgowda , Fukumoto Jutaro , Saji Smita , Hernandez‐Cuervo Helena , Alleyn Matthew , Lockey Richard F , Kolliputi Narasaiah , Galam Lakshmi
Published: June 2021

Rationale

Acute respiratory distress syndrome (ARDS) is associated with fluid filled lungs and hypoxia. Thus, ARDS patients are placed on supplemental oxygen; however, hyperoxia can damage the lungs, cause mitochondrial dysfunction, and ultimately lead to acute lung injury in animal models of ARDS. Mitochondrial aldehyde dehydrogenase 2 (Aldh2) metabolizes dangerous, reactive products, such as 4‐hydroxynonenal, that otherwise causes oxidative stress. The focus of this research is to determine if Alda‐1, an Aldh2 activator enhances the activity of Aldh2, thus alleviating hyperoxia damage by preventing mitochondrial dysfunction and reducing immune cell infiltration. It also determined if administration of Alda‐1 recovers mitochondrial dynamics and reduces cytokine levels and immune cell infiltration.

Methods

C57BL/6 mice were exposed to hyperoxia for 48 hours with DMSO (Control) and Alda‐1 (20 mM) via alzet pumps on the dorsal side of mice. The mice were divided into four groups: normoxia + DMSO (room air), normoxia + Alda‐1, hyperoxia + DMSO, and hyperoxia + Alda‐1. The lungs were harvested and the bronchoalveolar lavage (BAL) fluid was collected and analyzed by immunoblot and cytokine levels.

Results

Immunoblot analysis of lung lysate demonstrates that mice treated with Alda‐1, compared to mice treated with DMSO, during hyperoxia have decreased levels of OPA1 and Drp1, indicating that Alda‐1 shields against oxidative stress for proteins associated with mitochondrial dynamics. Moreover, the BAL fluid analysis reveals less infiltration of macrophages in mice treated with Alda‐1 versus mice treated with DMSO alone. These results indicate that activation of Aldh2 via Alda‐1 is protective against hyperoxia‐induced acute lung injury and may be a viable therapeutic agent for ARDS.

Conclusion

These findings suggest that Alda‐1, an Aldh2 activator, maintains mitochondrial homeostasis and reduces immune cell infiltration in this mouse model of ARDS and may be a novel therapeutic agent in ARDS.

Support or Funding Information

This work is supported by NIH RO1 grant (HL105932) to N.K. and AHA grant (#17SDG32780002) to L.G.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Requirement of Sterol Regulatory Element‐Binding Protein Pathway in Pancreatic Ductal Adenocarcinoma
Authors: Myers Stephanie , McGuire Meredith , Shao Wei , Ewachiw Ted , Rasheed Zeshaan , Matsui William , Espenshade Peter
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature, PDAC tumor cells have a high demand for lipid synthesis. However, this tumor is known to be poorly vascularized and nestled within a hypoxic environment. As lipid synthesis is a highly oxygen‐consumptive process, neoplastic cells are challenged with meeting the demand for lipids. Cells, including cancer cells, respond to lipid conditions through sterol regulatory element‐binding proteins (SREBPs). SREBPs are master regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling to regulate lipid synthesis. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. Here, we acquired four patient‐derived pancreatic adenocarcinoma cell lines, knocked out SCAP, then followed with a rescue. We performed functional growth assays using both lipid‐poor and lipid‐rich conditions as well as subcutaneous and orthotopic xenografts in nude mice. In lipid‐poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wildtype or SCAP rescued cells. In subcutaneous tumor xenografts, SCAP knockout cells exhibited reduced tumor volume in 3 out of 4 cell lines when compared to parent wildtype tumor cells. Similarly, SCAP knockout cells grew poorly in pancreatic orthotopic xenograft models. Our results demonstrate that loss of SCAP in PDAC tumor cells alters growth both in vitro and in vivo. These findings suggest SCAP may be useful as a therapeutic target in combating PDAC.

Support or Funding Information

NIH T32 OD011089 (SM); Allegheny Health Network Cancer Grant (PJE); Sol Goldman Pancreatic Cancer Research Center Grant from Sidney Kimmel Comprehensive Center (PJE); NIH F31LB131185 (MM)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Glucose Metabolism in Medulloblastoma
Authors: Telang Sucheta , Meiman Evan , Burlison Joseph
Published: June 2021

Medulloblastoma is the most common malignant brain tumor of childhood. Although current treatment approaches for this cancer have improved overall survival, they leave devastating and lifelong sequelae and the mortality remains high. Novel targeted therapies are therefore crucial to improve survival in these children and decrease treatment‐related toxicity.

Medulloblastoma development is largely driven by deregulation of normal cerebellar proliferation wherein the sonic hedgehog (Shh) pathway plays a critical role. Activating mutations of Shh genes are frequent in medulloblastoma and promote their rapid growth. In addition, highly aggressive Shh‐active medulloblastomas also exhibit mutations of the Ras/MAPK pathway and increased HIF‐1α, which are observed to stimulate glycolysis by activating the 6‐phosphofructo‐2‐kinase/fructose‐2,6 bisphosphatase family of enzymes (PFKFB1‐4). The PFKFBs produce fructose‐2,6‐bisphosphate (F26BP), a potent activator of 6‐phosphofructo‐1‐kinase, a rate‐limiting step in glycolysis. Recent data indicate that the PFKFB4 family member is increased in multiple human tumors and cancer lines, required for cell survival in aggressive brain tumors and strongly induced by hypoxia. We have found that the PFKFB4 enzyme was highly expressed in a series of patient‐derived medulloblastomas and correlated significantly with expression of the Shh pathway effector Smoothened (SMO) and GLI1. Through HIF‐1α upregulation, hypoxia was observed to strongly induce PFKFB4 expression in Shh‐active medulloblastoma cells and silencing PFKFB4 in these cells markedly suppressed F26BP and glycolytic flux to lactate and significantly decreased cell survival and proliferation, more prominently in hypoxia, indicating that PFKFB4 may be preferentially required for survival and growth under hypoxia. In addition, we found that co‐silencing PFKFB4 with SMO or GLI1 markedly decreased cell survival. We have recently developed a novel selective small molecule inhibitor of PFKFB4 and examined the effects of inhibiting PFKFB4 and the Shh pathway simultaneously. We found that simultaneous administration of the PFKFB4 inhibitor and inhibitors of the Shh pathway synergistically decreased Shh‐active medulloblastoma cell viability. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option against medulloblastoma and strongly support the further examination of simultaneous inhibition of PFKFB4 and the Shh pathway as a treatment strategy in medulloblastoma.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained Polyamine Depletion for the Treatment of Pancreatic Ductal Adenocarcinoma
Authors: Dobrovolskaite Aiste , Altomare Deborah , Phanstiel Otto
Published: June 2021

Of all human tissues, the human pancreas has the highest level of the polyamine spermidine. Pancreatic cancers use these high levels to drive their growth. Indeed, polyamine metabolism plays critical roles in many cellular processes including transcription, translation, and chromatin remodeling. Therefore, therapies, which deplete the intracellular pools of the native polyamines putrescine, spermidine, and spermine, result in cell growth arrest. Inhibitors have been designed to inhibit the rate limiting enzyme in polyamine biosynthesis, i.e., ornithine decarboxylase (ODC). Indeed, the ODC inhibitor difluoromethylornithine (DFMO), has a forty year history in the clinic with mixed results as an oncology therapeutic. Unfortunately, DFMO‐treated cancer cells often respond by increasing polyamine import to restore their depleted polyamine pools. Therefore, in order to establish sustained intracellular polyamine depletion, a combination therapy of DFMO and a polyamine transport inhibitor (PTI) is required.

New polyamine transport inhibitors are needed because all existing PTIs are polyamine‐based and can give rise to off‐target effects. To address this need, we have developed new PTI agents predicated upon a benzoic acid scaffold. This report describes our development of these new agents and their use to inhibit the uptake of radiolabeled spermidine and block the rescue of DFMO‐treated pancreatic cancer cells by exogenous spermidine. Since pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic cancer, we have focused on this cancer type in our studies. A success here could lead to new therapies for the world's most deadly cancer, which currently has a five year survival of only 8%.

Support or Funding Information

2016 Florida Translational Research Program and the 2018 Bankhead‐Coley Cancer Research Program Award # 8BC05.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of lipid accumulation in monocytes and macrophages by cyclodextrin‐based nanocarriers for alpha‐tocopheryl phosphate
Authors: Zingg Jean‐Marc , Stamatiou Christina , Daunert Sylvia
Published: June 2021

Vitamin E can modulate the CD36/FAT scavenger receptor/fatty acids transporter activity and affect lipid accumulation in monocytes and macrophages, important events for inflammation, lipotoxicity and foam cells formation during atherosclerosis. Similar to that, cyclodextrins (CD) can sequester and trigger export of lipids and influence cholesterol‐mediated signaling and gene expression leading to macrophages reprogramming and lipid lowering effects in a number of disease models. We have now combined the phosphorylated vitamin E analogue (alpha‐tocopheryl phosphate, αTP) and beta‐cyclodextrin (βCD) in a novel formulation to evaluate its regulatory effects and mechanisms of action on lipid accumulation in human monocytes/macrophages. In human THP‐1 monocytes and macrophages, the αTP/βCD complex not only increased the cellular delivery of αTP, but also reduced lipid accumulation by reducing CD36/FAT surface exposition and/or by enhancing the removal of cholesterol from the cells/membranes. A comparable activity was observed with higher order complexes of αTP/βCD with three types of modified starches or chitosan that may be useful as protective vehicle to increase oral, dermal or ocular delivery, or act as deposit‐enhancer in mucosa for delivery across epithelia. Moreover, preliminary results suggest that the αTP/βCD complex can influence signal transduction and gene expression pathways centrally involved in regulating cellular lipid homeostasis and inflammation. Thus, by acting as a Dual Action Lipid‐lowering Complex (DALC), αTP/βCD may not only increase the delivery of αTP and reduce cellular lipid accumulation by influencing CD36‐mediated signaling and gene expression relevant for lipid homeostasis, inflammation and cellular senescence, but also by stimulating βCD‐mediated lipid removal and macrophage reprogramming. In vivo, similar or better reductions of cellular lipids/cholesterol may be achieved possibly at lower concentrations as result of increased bioavailability and stability of αTP and of enhanced βCD‐mediated lipid exchange and macrophages reprogramming relevant not only for inflammation and atherosclerosis, but also for non‐alcoholic steatohepatitis (NASH), tumorigenesis and Parkinson's Disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of n‐3 fatty acid status of pregnant women in Zambia with relation to HIV infection
Authors: Majid Sonoor , Kisling Sophia , Allen James , Wood Charles , Chunda Catherine , DiRusso Concetta
Published: June 2021

Malnutrition and human immunodeficiency virus (HIV) are independent risk factors for impairment of fetal and early childhood development, altered brain structure, and reduced cognitive function. Docosahexaenoic acid (DHA (C22:6cisΔ 4,7,10,13,16,19 ω3)) is a fatty acid that is abundant in the brain, retina, and cerebral cortex. Additionally, DHA‐derived eicosanoids function in the resolution of inflammation. Thus, there it is hypothesized to be a link between DHA status, brain function and inflammation that may be critical in HIV positive pregnant women. Brain development and DHA incorporation into neuronal tissues occur largely during the last trimester of pregnancy to 24 months after birth. DHA may be acquired either through de novo synthesis from linolenic acid (LN (C18:3 cisΔ 9,12,15 ω3)) or from the diet. The present study was undertaken to evaluate DHA status in terms of dietary intake and plasma levels in pregnant women in Zambia, a country where food insecurity and HIV infection are highly prevalent. Initially, 113 women were enrolled in this study at the antenatal clinic of the Women and Children's Hospital in Lusaka, Zambia. The participants completed a health and dietary questionnaire and then a fasting blood sample was acquired. Ultimately, 45 HIV seronegative and 46 HIV seropositive women were included in the study cohort. Pre‐pregnancy BMI had a mean of 22 and ranged from 17.9 to 24.9, thus none of the women were clinically underweight. Gestational age ranged from 20–29 weeks with an average of 22. Blood lipids were measured to quantitate and compare triglyceride, lipoprotein and total cholesterol, and fatty acid levels. Total fatty acid profiles were assessed using gas chromatography/mass spectrometry (GC/MS). Essentially all participants reported eating fish on a regular basis averaging 2‐times per week and most (80%) consumed the small sardine‐like fresh water fish Kapenta, that is known to be relatively high in DHA. However, plasma fatty acid profiles indicated a high ω6 to ω3 ratio and low LN, EPA and DHA levels overall. There were no significant differences in these values between HIV seropositive and seronegative participants. Therefore, we propose DHA supplementation is warranted to raise blood DHA levels during pregnancy in Zambia to address both child development and the mother's health.

Support or Funding Information

World Health Organization (WHO)

Undergraduate Creative Activities and Research Experience (UCARE)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Nicotine on Adipocyte Production of Resistin and NF‐Kβ Translocation
Authors: Peter Jennica Renae
Published: June 2021

The relationship between nicotine and inflammation is complex and understanding the role of nicotine in inflammation could hold the key to treating many inflammatory diseases. Resistin is a pro‐inflammatory adipokine that is associated with inflammatory diseases, including ulcerative colitis. Anecdotal evidence indicates that patients with ulcerative colitis can achieve symptom relief by smoking commercial cigarettes, and this has prompted human and murine studies. We hypothesized that the mechanism through which nicotine affects adipocytes is a decrease in resistin expression via modification of NFKb activation. 3T3‐L1 mouse fibroblasts were differentiated into adipocytes and exposed to 10uM nicotine for 26 hours. We found that resistin release was decreased in nicotine‐treated adipocytes (n=23, μ=57.193 pg/ml, s=29.546) when compared to the control (n=8, μ=4,933 pg/ml, s=8.068), nearly a 100‐fold decrease. We conclude that nicotine reduces the secretion of resistin from adipocytes and that this may be the immunosuppressive mechanism underlying nicotine effects in the treatment of chronic inflammatory disease.

Support or Funding Information

Fort Lewis College Biology

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H‐Ras Signaling Mediates Microglia Proliferation Contributing to Neuropathology in INCL Mice
Authors: Sadhukhan Tamal , Bagh Maria , Appu Abhilash , Mondal Avisek , Kong Eryan , Liang Yinming , Zhang Zhongjian , Mukherjee Anil B
Published: June 2021

In virtually all neurodegenerative diseases, including infantile neuronal ceroid lipofuscinosis (INCL), neuronal death follows microglial proliferation. Proliferating microglia release toxic cytokines, which are suggested to cause neuroinflammation and progressive death of viable neurons contributing to neurodegeneration. Despite this knowledge, the molecular mechanism(s) underlying microglial proliferation in neurodegenerative diseases remains poorly understood. INCL is a neurodegenerative lysosomal storage disease, caused by inactivating mutations in the CLN1 gene encoding palmitoyl‐protein thioesterase‐1 (PPT1). PPT1 depalmitoylates S‐acylated proteins (constitutes of ceroid), essential for their degradation by lysosomal hydrolases. Thus, PPT1‐deficiency causes lysosomal ceroid accumulation leading to INCL. S‐palmitoylation (also called S‐acylation) is a reversible post‐translational lipid‐modification, which regulates the function of many proteins, especially in the brain. In humans, a family of 23 palmitoyl‐acyltransferases (PATs), containing a DHHC (Asp‐His‐His‐Cys) motif (DHHC‐PATs), catalyze S‐palmitoylation. In contract, thioesterases, localized to the cytosol or to the lysosome, depalmitoylate S‐acylated proteins. We previously reported that cytosolic thioesterase, APT1, requires S‐palmitoylation for membrane‐anchorage and catalyzes its own depalmitoylation to promote dynamic palmitoylation (palmitoylation‐depalmitoylation), essential for its cytosol‐membrane shuttling. Notably, proto‐oncogene H‐Ras product also requires dynamic S‐palmitoylation for cytosol‐membrane shuttling for signaling. Moreover, S‐palmitoylated H‐Ras is a substrate of APT1. Despite the demonstration that APT1 self‐catalyzes depalmitoylation, the DHHC‐PAT(s) that S‐palmitoylates APT1, remains unknown. Here we report that DHHC5 and DHHC23 catalyze S‐palmitoylation of APT1 promoting its membrane‐anchorage. Moreover, in Cln1−/− mice, which mimic INCL, the levels of S‐palmitoylated H‐Ras in the brain are significantly higher compared with those in WT controls. Unexpectedly, we found that the levels of DHHC5 and DHHC23 in Cln1−/− mice are significantly reduced. These results may suggest Ppt1‐Apt1 cross‐talk is mediated via regulation of these two DHHCs. We propose that crosstalk between Apt1 and Ppt1 elevates membrane‐anchored, S‐palmitoylated‐H‐Ras, which transduces signals to stimulate astroglia proliferation contributing to rapid progression of neurodegeneration in Cln1−/− mice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of cholesterol trafficking in BACE‐1 and APP co‐localization
Authors: Levitt Claire Hoa , Tarren Anna , Cluett Edward
Published: June 2021

Beta amyloid (Aβ) peptides play a central role in the pathogenesis of Alzheimer's disease (AD). Aβ peptides are derived from the processing of amyloid precursor protein (APP). This protein undergoes extensive processing involving two different pathways that produces several fragments. The α‐secretase pathway produces normal peptides. The pathogenic form of Aβ is derived from a cleavage product generated by beta‐site APP Cleaving Enzyme 1 (BACE‐1) that is then cleaved by γ‐secretase. Although the interaction of APP and BACE‐1 is critical for understanding how AD occurs, it is not clear how or where this occurs. Cholesterol has been identified as a risk factor in AD, and in vitro studies suggest that increased cholesterol levels promote misprocessing of APP. Our lab found that phospholipase A2 inhibitors, which inhibit the formation of membrane tubules, caused the accumulation of cholesterol in the recycling endosome in HeLa cells grown in moderate levels of LDL. APP also co‐localized to this compartment, and immunoblotting showed that the processing of APP and the association of APP fragments with cholesterol‐rich domains was altered by treatment with ONO‐RS‐082, a PLA2 inhibitor. However, BACE‐1 did not co‐localize with APP under these conditions. Surprisingly, in SY‐SH5Y cells co‐localization with BACE‐1 and APP did occur when cells were grown in low levels of LDL, which promote the synthesis of cholesterol. Using immunofluorescence/confocal microscopy and immunoblotting, we are confirming these results and extending this research by investigating the effects of statins or increased LDL on the localization and processing of these proteins.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.