Downregulation Of Transporters Research Articles

Transcriptomics of SGLT2-positive early proximal tubule segments in mice: response to type 1 diabetes, SGLT1/2 inhibition, or GLP1 receptor agonism.

SGLT2 inhibitors (SGLT2i) and GLP1 receptor (GLP1R) agonists have kidney protective effects. To better understand their molecular effects, RNA sequencing was performed in SGLT2-positive proximal tubule segments isolated by immunostaining-guided laser capture microdissection. Male adult DBA wild-type (WT) and littermate diabetic Akita mice ± Sglt1 knockout (Sglt1-KO) were given vehicle or SGLT2i dapagliflozin (dapa; 10 mg/kg diet) for 2 wk, and other Akita mice received GLP1R agonist semaglutide [sema; 3 nmol/(kg body wt·day), sc]. Dapa (254 ± 11 mg/dL) and Sglt1-KO (367 ± 11 mg/dL) but not sema (407 ± 44 mg/dL) significantly reduced hyperglycemia in Akita mice (480 ± 33 mg/dL). The 20,748 detected annotated protein-coding genes included robust enrichment of S1-segment marker genes. Akita showed 198 (∼1%) differentially expressed genes versus WT (DEGs; adjusted P ≤ 0.1), including downregulation of anionic transport, unsaturated fatty acid, and carboxylic acid metabolism. Dapa changed only two genes in WT but restored 43% of DEGs in Akita, including upregulation of the lipid metabolic pathway, carboxylic acid metabolism, and organic anion transport. In Akita, sema restored ∼10% of DEGs, and Sglt1-KO and dapa were synergistic (restored ∼61%), possibly involving additive blood glucose effects (193 ± 15 mg/dL). Targeted analysis of transporters and channels (t test, P < 0.05) revealed that ∼10% of 526 detectable transporters and channels were downregulated by Akita, with ∼60% restored by dapa. Dapa, dapa + Sglt1-KO, and sema also altered Akita-insensitive genes. Among DEGs in Akita, ∼30% were unresponsive to any treatment, indicating potential new targets. In conclusion, SGLT2i restored transcription for multiple metabolic pathways and transporters in SGLT2-positive proximal tubule segments in diabetic mice, with a smaller effect also observed for GLP1R agonism.NEW & NOTEWORTHY SGLT2 inhibitors and GLP1 receptor agonists have kidney protective effects. By combining immunostaining-guided laser capture microdissection and RNA sequencing, the study established how the gene expression profile changes in SGLT2-positive proximal tubule cells in response to type 1 Akita diabetes and to pharmacological intervention by SGLT2 inhibition or GLP1R agonism and genetic deletion of SGLT1. The data also indicate genes unresponsive to those treatments that may include new therapeutical candidates.

Alleviation of drought stress in tomato by foliar application of seafood waste extract

To manage the adverse effects of garbage pollution and avoid using chemicals, a natural extract of seafood shells was obtained and explored for its beneficial role. Physical characterization highlighted that its active compounds correspond to chitin and its derivative, chitosan. The ability of the extracted biostimulant to foster tomato tolerance was tested on drought-stressed plants. Along with changes in morphological parameters, the accumulation of chlorophyll and carotenoids was improved. The biostimulant also mediates the accumulation of osmoprotectants and an increased leaf water content. Furthermore, the biostimulant effectively promotes tolerance by increasing drought-stress SIERF84 Transcription factor and decreasing both SIARF4 and SlWRKY81 transcript levels, which in turn, mediates stomatal closure. In addition, the up-regulation of key genes related to NO3− uptake (NTR1.1/2) and assimilation (NR) coupled with the downregulation of ammonium transporters’ genes (AMT1.1/2), allowed the uptake of NO3− over NH4+ in the tolerant genotype which is likely to be associated with drought tolerance. Overall, the biostimulant was effective in alleviating water stress and showed similar effects to commercial chitosan. Besides the benefits of a circular economy framework, this biostimulant-based approach is innovative to promote a sustainable eco-agriculture, in the face of persistent water scarcity.

NIMG-19. DEUTERIUM METABOLIC IMAGING INTERROGATES THE HISTONE H3K27M MUTATION AND PROVIDES AN EARLY READOUT OF RESPONSE TO THERAPY IN DIFFUSE MIDLINE GLIOMAS

Abstract Diffuse midline gliomas (DMGs) are lethal primary brain tumors in children that are driven by recurrent lysine 27 to methionine mutations in histone H3 (H3K27M). Radiation is standard of care and the imipridones ONC201 and ONC206 have emerged as promising therapies for DMG patients. Although magnetic resonance imaging (MRI) is the mainstay for patient management, it does not reliably assess response to therapy. Therefore, the goal of this study was to identify metabolic alterations induced by H3K27M that can be leveraged for non-invasive metabolic imaging of DMGs. Using patient-derived (BT245, SF8628, SF7761, SU-DIPG-6, DIPG7, SU-DIPG-13) and syngeneic (26-B7, 26-C2) models, we show that the H3K27M mutation upregulates glucose metabolism via glycolysis to lactate. Silencing the H3K27M mutation reduces glycolytic capacity and depletes lactate, an effect that is associated with downregulation of rate-limiting glycolytic enzymes and transporters (SLC2A1, HK2, PFKFB3, PGK1, SLC16A3). Deuterium metabolic imaging (DMI) is a novel, clinical stage method of tracing glucose metabolism in vivo. Our studies show that lactate production from [6,6-2H]-glucose is spatially localized to tumor vs. normal brain in mice bearing intracranial BT245, SF8628 or 26-B7 xenografts at clinical magnetic field strength (3T). Lactate production from [6,6’-2H]-glucose is reduced in patient-derived (BT245, SF8628, SF7761) or syngeneic (26-B7) cells treated with radiation, ONC201 or ONC206. Importantly, lactate production is reduced at an early timepoint (5 days) following treatment with ONC206, when changes cannot be detected by MRI, in mice bearing intracranial BT245 or SF8628 xenografts. Collectively, our studies link the H3K27M mutation with elevated glycolysis and identify [6,6’-2H]-glucose as a safe, orally administered contrast agent for visualizing the metabolically active lesion and for imaging early response to therapy in DMGs in vivo. Clinical translation of our studies will provide physicians with a much-needed tool to determine whether DMG patients are responding to standard and experimental therapies under development.

Development and implementation of a simulated microgravity setup for edible cyanobacteria

Regenerative life support systems for space crews recycle waste into water, food, and oxygen using different organisms. The European Space Agency’s MELiSSA program uses the cyanobacterium Limnospira indica PCC8005 for air revitalization and food production. Before space use, components’ compatibility with reduced gravity was tested. This study introduced a ground analog for microgravity experiments with oxygenic cyanobacteria under continuous illumination, using a random positioning machine (RPM) setup. L. indica PCC8005 grew slower under low-shear simulated microgravity, with proteome analysis revealing downregulation of ribosomal proteins, glutamine synthase, and nitrate uptake transporters, and upregulation of gas vesicle, photosystem I and II, and carboxysome proteins. Results suggested inhibition due to high oxygen partial pressure, causing carbon limitation when cultivated in low-shear simulated microgravity. A thicker stagnant fluid boundary layer reducing oxygen release in simulated microgravity was observed. These findings validate this RPM setup for testing the effects of non-terrestrial gravity on photosynthetic microorganisms.

Comparison of Glucose Metabolizing Properties of Enterobacterial Probiotic Strains In Vitro.

Before the absorption in the intestine, glucose encounters gut bacteria, which may serve as a barrier against hyperglycemia by metabolizing glucose. In the present study, we compared the capacity of enterobacterial strains to lower glucose levels in an in vitro model of nutrient-induced bacterial growth. Two probiotic strains, Hafnia alvei HA4597 (H. alvei) and Escherichia coli (E. coli) Nissle 1917, as well as E. coli K12, were studied. To mimic bacterial growth in the gut, a planktonic culture was supplemented twice daily by the Luria Bertani milieu with or without 0.5% glucose. Repeated nutrient provision resulted in the incremental growth of bacteria. However, in the presence of glucose, the maximal growth of both strains of E. coli but not of H. alvei was inhibited. When glucose was added to the culture medium, a continuous decrease in its concentration was observed during each feeding phase. At its highest density, H. alvei displayed more efficient glucose consumption accompanied by a more pronounced downregulation of glucose transporters' expression than E. coli K12. Thus, the study reveals that the probiotic strain H. alvei HA4597 is more resilient to maintain its growth than E. coli in the presence of 0.5% glucose accompanied by more efficient glucose consumption. This experimental approach offers a new strategy for the identification of probiotics with increased glucose metabolizing capacities potentially useful for the prevention and co-treatment of type 2 diabetes.

Unraveling the Complex Role of Zinc, Boron, Chromium, and Selenium in the Pathogenesis of Diabetes Mellitus: A Review

Trace elements are micronutrient components that are only required in small amounts but are critical for the biological functions of many human body tissues. Studies in multiple settings found significant connections between diabetic mellitus (DM) and trace elements caused by disturbances of overlapping cellular metabolic systems. Zinc, boron, chromium, and selenium at either extremely high or low levels could elicit some alteration in cellular metabolism. These lead to the development of DM. The changes include 1) the disturbance in the efficient release of insulin secretory granules, 2) the production of proinflammatory cytokines and oxidative stress state, 3) the failure of insulin signalling pathway, and 4) the reduction of glucose tissue uptake secondary to the downregulation of glucose transporters. Both significantly high and low concentrations have been linked to the development of insulin resistance. Nevertheless, conflicting evidence makes their optimum nutritional levels difficult to establish. For example, lead at a trace concentration may accelerate the development of insulin resistance. The purpose of this review is to emphasise the metabolic role of the 4 trace elements and their influence on the pathogenesis of diabetes when body levels are below optimal. Understanding the consequences of these elements could pave the way for therapeutic possibilities and breakthroughs in personalised DM management.

Cholesterol Dietary Intake and Tumor Cell Homeostasis Drive Early Epithelial Tumorigenesis: A Potential Modelization of Early Prostate Tumorigenesis.

Epidemiological studies point to cholesterol as a possible key factor for both prostate cancer incidence and progression. It could represent a targetable metabolite as the most aggressive tumors also appear to be sensitive to therapies designed to decrease hypercholesterolemia, such as statins. However, it remains unknown whether and how cholesterol, through its dietary uptake and its metabolism, could be important for early tumorigenesis. Oncogene clonal induction in the Drosophila melanogaster accessory gland allows us to reproduce tumorigenesis from initiation to early progression, where tumor cells undergo basal extrusion to form extra-epithelial tumors. Here we show that these tumors accumulate lipids, and especially esterified cholesterol, as in human late carcinogenesis. Interestingly, a high-cholesterol diet has a limited effect on accessory gland tumorigenesis. On the contrary, cell-specific downregulation of cholesterol uptake, intracellular transport, or metabolic response impairs the formation of such tumors. Furthermore, in this context, a high-cholesterol diet suppresses this impairment. Interestingly, expression data from primary prostate cancer tissues indicate an early signature of redirection from cholesterol de novo synthesis to uptake. Taken together, these results reveal that during early tumorigenesis, tumor cells strongly increase their uptake and use of dietary cholesterol to specifically promote the step of basal extrusion. Hence, these results suggest the mechanism by which a reduction in dietary cholesterol could lower the risk and slow down the progression of prostate cancer.

Extracellular Self-DNA Effects on Yeast Cell Cycle and Transcriptome during Batch Growth.

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.

Dietary Caffeine and Brain Dopaminergic Function in Parkinson Disease.

This study was undertaken to investigate the effects of dietary caffeine intake on striatal dopamine function and clinical symptoms in Parkinson disease in a cross-sectional and longitudinal setting. One hundred sixty-three early Parkinson disease patients and 40 healthy controls were investigated with [123I]FP-CIT single photon emission computed tomography, and striatal dopamine transporter binding was evaluated in association with the level of daily coffee consumption and clinical measures. After a median interval of 6.1 years, 44 patients with various caffeine consumption levels underwent clinical and imaging reexamination including blood caffeine metabolite profiling. Unmedicated early Parkinson disease patients with high coffee consumption had 8.3 to 15.4% lower dopamine transporter binding in all studied striatal regions than low consumers, after accounting for age, sex, and motor symptom severity. Higher caffeine consumption was further associated with a progressive decline in striatal binding over time. No significant effects of caffeine on motor function were observed. Blood analyses demonstrated a positive correlation between caffeine metabolites after recent caffeine intake and dopamine transporter binding in the ipsilateral putamen. Chronic caffeine intake prompts compensatory and cumulative dopamine transporter downregulation, consistent with caffeine's reported risk reduction in Parkinson disease. However, this decline does not manifest in symptom changes. Transiently increased dopamine transporter binding after recent caffeine intake has implications for dopaminergic imaging guidelines. ANN NEUROL 2024;96:262-275.

Zinc transporters expression profile in professional handball players supplemented with zinc

IntroductionZinc (Zn) deficiency has been described not only on general human health but also within the sports context –as negatively affecting performance–. Thus, Zn status assessment is of great interest for athletes, especially in order to correct deficiency states of this mineral. ObjectiveThe overall objective of this work was to assess Zn status in professional handball players during the competitive period (through plasma levels, dietary intake and gene expression of the Zn transporters), as well as to determine the effect of Zn supplementation. MethodsA total of twenty-two participants were recruited, –twelve belonged to the Control Group (CG) and ten male handball players comprised the experimental group (ATH-G)–, being monitored over a 2-month period with 2 evaluation moments: baseline (i.e., initial conditions) and follow-up (i.e., after 8 weeks of training and competition). Zn intake, plasma Zn levels, and gene expression of Zn transporters were obtained. ResultsPlasma Zn levels were higher in ATH-G than in CG at the end of Zn intervention (p ≤ 0.010). Moreover, differences in the gene expression profile of Zn transporters were observed in ATH-G –with the down-regulation of several Zn transporters–, compared to the CG at baseline (p ≤ 0.05). Likewise, differences in the Zn transporters expression were observed in ATH-G at 8 weeks (all, p ≤ 0.001) –with ZnT2, ZnT5, ZIP3, ZIP5, ZIP11, ZIP13 and ZIP14 transporters being up-regulated–. ConclusionHandball players seemed to have different nutritional needs for Zn, with differences in the gene expression of Zn transporters compared to controls. Zn intervention in our athletes may have influenced the expression of Zn transporters, indicating a potential increase in Zn transporters expression to mobilize Zn at the cellular level at 8 weeks of Zn intervention.

Abstract PO3-08-07: Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers

Abstract Background: Breast cancer risk reducing drugs with proven efficacy have adverse side effects, significantly minimizing their uptake and impact. Further, they do not prevent ER- breast cancer. Effective alternative strategies with lower toxicity are needed. Previously, we have shown that licochalcone A (LicA) suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. These data led us to hypothesize that LicA creates a tumor preventive environment in the breast by reprogramming metabolism and antioxidant/anti-inflammatory responses in the breast leading to decreased proliferation and tumor suppression. We now report on the breast tumor preventive effects of LicA in xenograft models, its oral bioavailability, and its biologic effects on human breast microstructures from women at increased risk of breast cancer. Methods: We prepared microstructures from the fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing. Differentially expressed genes were identified and analyzed by gene ontology and pathway membership. The RNA-seq data was also utilized to conduct metabolism flux analysis. Combined enrichment scores &amp;gt; 4 and FDR &amp;lt; 0.05 was considered significant. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells. Xenograft models of MCF-7aro and MDA-MB-231 tumors were established in female nude mice and the animals treated for 28 days with vehicle or LicA (80 mg/kg.day, s.c.). We measured the rate of tumor growth. We also conducted a PK/PD study with oral LicA (100 mg/kg) in intact BALB/c female mice. Results: We observed significant (FDR &amp;lt; 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system, the major regulators of antioxidant pathways. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis and transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and anti-proliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cells at doses &amp;lt; 10 µM. LicA significantly reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) in vivo models (unpaired t-test with Welch’s correction for unequal variances). Promising serum and breast bioavailability, equivalent to low micromolar concentrations sufficient to show efficacy was demonstrated as well. Conclusion: Our data suggest that LicA is a good candidate for breast cancer prevention in both ER+ and ER- breast cancers through reprogramming metabolism and antioxidant pathways leading to decreased proliferation. We will study LicA in intraductal models of ER+ and ER- precancer lesions in immunocompetent mice and will monitor their progression to invasive breast cancer to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-07.

Amino Acid Uptake Limitations during Human Mesenchymal Stem Cell-Based Chondrogenesis.

A mino acids are the essential building blocks for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins requires the uptake of various essential/nonessential amino acids. Analyzing amino acid metabolism during chondrogenesis can help to relate tissue quality to amino acid metabolism under different conditions. In our study, we studied amino acid uptake/secretion using human mesenchymal stem cell (hMSC)-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acid uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized glycosaminoglycans and hydroxyproline content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results show that amino acid uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of human neonatal cartilage or adult cartilage. Furthermore, this deficiency was likely related to the downregulation of some key amino acid transporters in the cells. Such deficiency could be partially improved by increasing the amino acid availability in the chondrogenic medium by changing culture conditions.

Impaired astrocytic synaptic function by peripheral cholesterol metabolite 27-hydroxycholesterol.

Astrocytes represent the most abundant cell type in the brain, where they play critical roles in synaptic transmission, cognition, and behavior. Recent discoveries show astrocytes are involved in synaptic dysfunction during Alzheimer's disease (AD). AD patients have imbalanced cholesterol metabolism, demonstrated by high levels of side-chain oxidized cholesterol known as 27-hydroxycholesterol (27-OH). Evidence from our laboratory has shown that elevated 27-OH can abolish synaptic connectivity during neuromaturation, but its effect on astrocyte function is currently unclear. Our results suggest that elevated 27-OH decreases the astrocyte function in vivo in Cyp27Tg, a mouse model of brain oxysterol imbalance. Here, we report a downregulation of glutamate transporters in the hippocampus of CYP27Tg mice together with increased GFAP. GLT-1 downregulation was also observed when WT mice were fed with high-cholesterol diets. To study the relationship between astrocytes and neurons, we have developed a 3D co-culture system that allows all the cell types from mice embryos to differentiate in vitro. We report that our 3D co-cultures reproduce the effects of 27-OH observed in 2D neurons and in vivo. Moreover, we found novel degenerative effects in astrocytes that do not appear in 2D cultures, together with the downregulation of glutamate transporters GLT-1 and GLAST. We propose that this transporter dysregulation leads to neuronal hyperexcitability and synaptic dysfunction based on the effects of 27-OH on astrocytes. Taken together, these results report a new mechanism linking oxysterol imbalance in the brain and synaptic dysfunction through effects on astrocyte function.

Obesogenic diet in pregnancy disrupts placental iron handling and ferroptosis and stress signalling in association with fetal growth alterations

Obesity and gestational diabetes (GDM) impact fetal growth during pregnancy. Iron is an essential micronutrient needed for energy-intense feto-placental development, but if mis-handled can lead to oxidative stress and ferroptosis (iron-dependent cell death). In a mouse model showing maternal obesity and glucose intolerance, we investigated the association of materno-fetal iron handling and placental ferroptosis, oxidative damage and stress signalling activation with fetal growth. Female mice were fed a standard chow or high fat, high sugar (HFHS) diet during pregnancy and outcomes were measured at day (d)16 or d19 of pregnancy. In HFHS-fed mice, maternal hepcidin was reduced and iron status maintained (tissue iron levels) at both d16 and d19. However, fetal weight, placental iron transfer capacity, iron deposition, TFR1 expression and ERK2-mediated signalling were reduced and oxidative damage-related lipofuscin accumulation in the placenta was increased in HFHS-fed mice. At d19, whilst TFR1 remained decreased, fetal weight was normal and placental weight, iron content and iron transporter genes (Dmt1, Zip14, and Fpn1) were reduced in HFHS-fed mice. Furthermore, there was stress kinase activation (increased phosphorylated p38MAPK, total ERK and JNK) in the placenta from HFHS-fed mice at d19. In summary, a maternal HFHS diet during pregnancy impacts fetal growth trajectory in association with changes in placental iron handling, ferroptosis and stress signalling. Downregulation of placental iron transporters in HFHS mice may protect the fetus from excessive oxidative iron. These findings suggest a role for alterations in placental iron homeostasis in determining perinatal outcomes of pregnancies associated with GDM and/or maternal obesity.Graphical

Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

Abstract 7306: Licochalcone A is an excellent candidate for preventing luminal and non-luminal breast cancers

Abstract Breast cancer (BC) risk reducing drugs have minimal impact due to their adverse effects and low acceptance by risk-eligible women. Further, they are ineffective against hormone receptor negative (HR-) BC. Novel agents with reduced toxicity and sufficient efficacy that extends beyond HR+ BC are needed. We showed previously that licochalcone A (LicA) from licorice has antioxidant/anti-inflammatory effects, inhibits aromatase, and blocks estrogen genotoxic metabolism. We hypothesize that LicA prevents HR+ and HR- BC through reprogramming metabolism and antioxidant pathways. We prepared microstructures from the fresh tissue of contralateral unaffected breast of 6 postmenopausal women with unilateral BC. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing followed by differential gene expression, pathway identification, and metabolism flux analyses. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of HR- and HR+ pre-malignant and malignant, as well as BRCA mutated cells. Xenograft models of HR+ and HR- tumors were established in female nude mice followed by 28-day treatment with vehicle or LicA (80 mg/kg.day, s.c.) and monitoring the tumor growth. Using LC-MS/MS we measured LicA in the blood and various tissues of intact BALB/c female mice receiving oral LicA (100 mg/kg) and assessed PK using Phoenix Platform. We observed significant (combined enrichment scores &amp;gt; 4 and FDR &amp;lt; 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis, transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and antiproliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cell lines at doses &amp;lt;10 µM. It reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) xenograft models. Oral bioavailability in serum and breast was in the low micromolar range and was sufficient to show efficacy.Our data suggest that LicA is an excellent candidate for both HR+ and HR- BC prevention by reprogramming metabolism and antioxidant pathways leading to decreased proliferation. Our next study will test an optimized oral formulation of LicA in intraductal models of HR+ and HR- precancer lesions in immunocompetent mice to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth T. Bartom, Sriram Chandrasekaran, Ruohui Chen, Jeremy J. Johnson, Susan E. Clare, Seema A. Khan. Licochalcone A is an excellent candidate for preventing luminal and non-luminal breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7306.

Abstract 5750: Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer

Abstract Introduction: Treatment choices for early-stage prostate cancer include mostly watchful waiting, radical prostatectomy, or radiation therapy, whereas for advanced cases hormone therapy followed by chemo/radiotherapy are the preferred options, despite their notable side effects. There is a need for more effective therapy with lower side effects, especially for advanced scenarios. Unlike normal epithelial prostate cells, cancerous ones show reduced zinc (Zn) levels due to ZIP1 zinc transporter downregulation. Zn deficiency allows m-aconitase to remain active, making prostate cancer cells energy efficient and grow. High levels of Zn are needed to inhibit m-aconitase, the enzyme responsible for the first reaction in the Krebs cycle. This study aims to induce energy starvation and cell death of prostate cancer cells by inhibiting m-aconitase using the prostate-specific membrane antigen (PSMA)-targeting liposomal zinc. Methods: Liposomes were prepared by standard reverse phase evaporation method with DSPC, Cholesterol and DSPE-mPEG(2000) lipids. Zinc chloride was used to load Zn+2 into liposome following passive loading method. PSMA-expressing (PC3-PIP) and PSMA-null (PC3-LUC) human prostate cancer cells were used for in vitro studies. In the cell uptake study, cells were incubated with Dil-dye labeled liposomes for 2 hours before analyzing them by microscopy and flow cytometry. For the uptake inhibition study, cells were pre-incubated with PSMA-specific peptide for 2 hours before incubation with liposomes. Cytotoxicity studies were performed with PSMA-targeting Zn-loaded liposomes in PC3-PIP cells. Results: We conjugated the PSMA-specific peptide and control peptide (ASP3) with DSPE-PEG(3400)-NHS, and used them at various amounts (1 to 4 mol% of total lipid) to prepare Zn-loaded PSMA-targeting liposomes (TL) and control liposomes (CL), respectively. The average hydrodynamic diameter of all the liposomes was 120 to 130 nm, and zeta potential ranged from -16 to -12 mV. The encapsulation efficiency and loading capacity of Zn+2 were 0.6% and 8.5%, respectively. Cellular uptake studies showed a higher accumulation of PSMA-targeting liposome (TL) compared to non-targeting liposome (CL) in PSMA-expressing PC3 cells, whereas both liposomes showed low uptake in PSMA non-expressing cell. Furthermore, pre-treatment of PSMA-expressing cells with PSMA-specific peptide reduced the uptake of TL, indicating that the uptake of TL liposome is PSMA dependent. Finally, cytotoxicity studies showed that Zn-loaded TL, induces cell death in the PSMA-expressing cells. Overall, we developed PSMA-targeting Zn-containing liposomes that selectively target and kill PSMA-expressing prostate cancer cells. Future work includes testing the effect of Zn-loaded TL on m-aconitase activity and testing of in vivo efficacy of the formulation in prostate cancer models. Citation Format: Sujan K. Mondal, Alexander L. Klibanov, Anna Moore. Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5750.

Sex-Dependent Changes to the Intestinal and Hepatic Abundance of Drug Transporters and Metabolizing Enzymes in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.

Stenotrophomonas strain CD2 reduces cadmium accumulation in Brassica rapa L.

IntroductionCadmium (Cd) is a highly toxic heavy metal which contaminates agricultural soils and is easily absorbed by plants. Brassica rapa L. is one of the most popular vegetables in China and is known to accumulate Cd in its roots and aerial tissues.MethodsA highly Cd-resistant bacterium (‘CD2’) was isolated and identified. Its ability to immobilize Cd(II) in medium was studied. Strain CD2 were added into Cd-polluted soil to ameliorate Cd accumulation in B. rapa. The underlying mechanisms of ‘CD2’ to reduce Cd accumulation in B. rapa. were analyzed by transcriptomics.Results and discussionStrain CD2 was classified as belonging to the genus Stenotrophomonas. Strain CD2 was found to be able to remove 0.1 mmol/L Cd(II) after 36 h by intracellular sequestration and by producing biofilm, exopolysaccharide, and H2S. When applied to Cd-contaminated soil, ‘CD2’ significantly increased the content of nonbioavailable Cd by 212.70%. Furthermore, ‘CD2’-inoculated B. rapa exhibited a 51.16% decrease in the Cd content of roots and a 55.56% decrease in the Cd content of aerial tissues. Transcriptome analysis identified 424 differentially expressed genes (DEGs) in the roots and 501 DEGs in the aerial tissues of uninoculated Cd-exposed plants. By comparison, 1107 DEGs were identified in the roots and 1721 DEGs were identified in the aerial tissues of ‘CD2’-inoculated Cd-exposed plants. In both treatment groups, genes related to vacuolar sequestration were upregulated, resulting in inhibited Cd transport. In addition, both catalase and glutathione transferase were induced in uninoculated plants, while the oxidative stress-related genes CPK and RBOH belonged to ‘plant-pathogen interactions’ were upregulated in ‘CD2’-inoculated plants. Moreover, inoculation with ‘CD2’ resulted in the enrichment of phenylpropane metabolism; cutin, suberine, and wax biosynthesis; and the AP2, Dof, WOX, Trihelix, B3, EIL, and M-type_MADS transcription factors; as well as the downregulation of zinc transporters and blue copper proteins. All of these changes likely contributed to the reduced Cd accumulation in ‘CD2’-inoculated B. rapa. The results of this study suggest that Stenotrophomonas sp. CD2 may prove to be a useful inoculant to prevent Cd accumulation in B. rapa.

Tetraploidy and Fe2O3 nanoparticles: dual strategy to reduce the Cd-induced toxicity in rice plants by ameliorating the oxidative stress and downregulation of metal transporters

Whole-genome doubling or polyploidy increases the plant tolerance ability against biotic and abiotic stress.