Mouse Embryo Fibroblast Cells Research Articles

Phytoactive essential oils-composed water-free organogels: development, characterization and proof of antibacterial activity

The aim of this work was the development and characterization of a waterless organogel formulation, based on phytoactives from essential oils known for their antimicrobial and anti-inflammatory activities. Lentisk tree (Pistacia lentiscus L.), yarrow (Achillea millefolium L.) and myrtle (Myrtus communis L.) grow in the mild climate of Euroasia and are receiving particular attention for their medicinal and aromatic value. Two distinct concentrations of selected essential oils (EOs) were tested, dispersed in organogels based on 3% (m/V) Carbopol 974P NF in PEG 400. Prior to formulation development, selected EOs were characterized by mass spectroscopy to identify the dominating bioactives. A total of 32 compounds were quantified for lentisk leaf EO, 42 for yarrow EO, and 26 for myrtle leaf EO. The best formulations were found for the highest concentration of EOs (4%, m/m) into Carbopol/PEG400 organogels. Organogels were produced by high-speed homogenization followed by microwave heating and showed acceptable pH between 5.8 – 6.0, being compatible with the skin, with an electrical conductivity highest for yarrow EO-based organogel (YIG4%, ∼ 64 mV), and the lowest for myrtle EO-based organogel (MIG4%, ∼ 48 mV). Texture analysis showed the highest adhesion properties for lentisk EO-based organogel (LIG 4%), attributed to its higher viscosity recorded for the two tested temperatures (25ºC vs 37ºC). Rheological analysis confirmed that organogels have pseudoplastic behaviour. The antibacterial activity was compared between plain EOs and EOs-based organogels, with the yarrow EO and its organogel (YIG 4%) depicting the highest activity against both tested bacterial strains (S. aureus and C. acnes). On the other hand, MIG 4% contributed for the highest cell proliferation rate tested in mouse embryo fibroblast cell (3T3-L1) cell line, whereas with LIG 4% the lowest viability value was recorded.

Cardioprotection and Suppression of Fibrosis by Diverse Cancer and Non-Cancer Cell Lines in a Murine Model of Duchenne Muscular Dystrophy.

The dynamic relationship between heart failure and cancer poses a dual challenge. While cardiac remodeling can promote cancer growth and metastasis, tumor development can ameliorate cardiac dysfunction and suppress fibrosis. However, the precise mechanism through which cancer influences the heart and fibrosis is yet to be uncovered. To further explore the interaction between heart failure and cancer, we used the MDX mouse model, which suffers from cardiac fibrosis and cardiac dysfunction. A previous study from our lab demonstrated that tumor growth improves cardiac dysfunction and dampens fibrosis in the heart and diaphragm muscles of MDX mice. We used breast Polyoma middle T (PyMT) and Lewis lung carcinoma (LLC) cancer cell lines that developed into large tumors. To explore whether the aggressiveness of the cancer cell line is crucial for the beneficial phenotype, we employed a PyMT breast cancer cell line lacking integrin β1, representing a less aggressive cell line compared to the original PyMT cells. In addition, we examined immortalized and primary MEF cells. The injection of integrin β1 KO PyMT cancer cells and Mouse Embryo Fibroblasts cells (MEF) resulted in the improvement of cardiac function and decreased fibrosis in the heart, diaphragm, and skeletal muscles of MDX mice. Collectively, our data demonstrate that the cancer line aggressiveness as well as primary MEF cells are sufficient to impose the beneficial phenotype. These discoveries present potential novel clinical therapeutic approaches with beneficial outcome for patients with fibrotic diseases and cardiac dysfunction that do not require tumor growth.

Abstract 1215: DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations

Abstract RAS mutations are found in 30% of human cancers and are characterized by hotspot mutations at codons G12, G13, and Q61. This results in constitutively activated RAS proteins. Despite recent advances in drug design and the emergence of covalent inhibitors, effectively targeting mutant RAS remains challenging. Our group previously identified the tumor suppressor protein DIRAS3, which directly binds Ras and inhibits its function by disrupting KRAS dimers/nanoclusters and blocking effector activation. DIRAS3 expression reduced cancer cell growth in pancreatic and ovarian cancers. To determine whether DIRAS3 plays a definitive role in inhibiting mutant KRAS, we comprehensively characterized DIRAS3's RAS-inhibitory effects using RASless mouse embryo fibroblast (MEF) cells as a model system lacking endogenous RAS isoforms. We ectopically expressed various KRAS mutations in these cells and then examined DIRAS3's effect on each KRAS mutant clone. We analyzed downstream RAS signaling, colony formation, cell viability, and toxicity. Our results showed that DIRAS3 significantly inhibited RAS-dependent colony formation in KRAS mutant (G12C, G12D, G12V, G13D, G12R, Q61R, Q61L) but not wild-type KRAS clones. However, when we added epidermal growth factor (EGF) to the medium, DIRAS3 expression inhibited wild-type KRAS activity, decreasing colony formation. This suggests that DIRAS3 selectively affects the active, GTP-bound form of KRAS and downstream signaling. Additionally, DIRAS3 expression decreased Erk1/2 phosphorylation in MEF cells with mutant KRAS, but not in the cells with a BRAFV600E mutation. This indicates that DIRAS3 specifically inhibits the KRAS-mediated MEK/ERK signaling, cell proliferation, and cytotoxicity. Moreover, to determine the critical region for DIRAS3's RAS-inhibitory function, we compared wild-type DIRAS3 and deletions (ΔNTE, ΔCTE, ΔNCTE). Our results showed both termini are required to effectively suppress clonogenic growth, emphasizing the requirement for DIRAS3 to anchor to the plasma membrane and interact with the RAS dimer interface to inhibit RAS-dependent tumor growth. In conclusion, our findings underscore the potent and specific inhibitory effects of DIRAS3 on RAS-driven oncogenesis, positioning it as a promising pan-RAS inhibitor for RAS-mutant cancers. Citation Format: Gamze Bildik, Junchen Liu, Weiqun Mao, Hailing Yang, John F. Hancock, Robert C. Bast, Zhen Lu. DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations [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 1215.

Optimization, Characterization, and Cytotoxic Study of Bio Cellulose by Acetobacter sp Strains to Engender Biodegradable Food Wrapper

Acetic acid bacteria synthesized microbial cellulose were isolated from various citrus fruits, enabled by increased cellulose production to develop a biodegradable polymer as a food wrapper. The objective of the research cynosures on the isolation, enrichment, identification, and optimization of bacteria that produce cellulose, characterization, and cytotoxic study of the obtained cellulose. Two highly effective cellulose producers, Acetobacter lovaniensis (A1) and Acetobacter fabarum (A2), were isolated based on their morphology, biochemical analysis, and 16s rRNA sequencing. Studies were conducted to optimize pH, temperature, inoculum size, nitrogen, and carbon sources. Strain A1 produced 0.715 g/100 ml, whereas A2 produced 0.856 g/100 ml of cellulose under optimum growth conditions. The characteristics of microbial cellulose were examined using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). A cytotoxicity study for the obtained cellulose has been conducted with mouse embryo fibroblast cells (3T3-L1) and showed 97% viability of cells with the lowest concentration of 12.5 µg/ml. These isolates could be employed in fermentation technology to produce cellulose polymer-based sustainable biodegradable food wrappers.

Wound healing and antidiabetic properties of green synthesized silver nanoparticles in 3T3-L1 mouse embryo fibroblast cells through 2-NBDG expression

The antibacterial and antifungal properties of silver and silver-based compounds have been recognized for quite some time. This study employed an extracellular method to produce silver nanoparticles (AgNPs) using the leaves of Barringtonia racemosa (B. racemosa). B. racemosa's aqueous leaf extract underwent a color change from grey to dark brown, which revealed the formation of AgNPs. UV spectrophotometry analysis confirmed the presence of silver with the maximum absorbance of 427 nm. FTIR analysis supported the existence of alcohols with the OH stretch and alkenes with the CC stretch. The existence of AgNPs with an average hydrodynamic diameter of 36.58 nm was confirmed using dynamic light scattering (DLS). Energy dispersive X-ray spectroscopy (EDX) confirmed the presence of silver. Additionally, the AgNPs showed spherical structure particles with the size distribution ranging from 10 to 17 nm with polydispersity, under transmission electron microscopy (TEM). Thermogravimetric analysis (TGA) findings revealed that AgNPs maintain good thermal stability even at high temperatures. A variety of bacterial and fungal pathogens were successfully inhibited by the AgNPs, with S. epidermidis (27 mm) showing the strongest antibacterial activity and a 20 mm inhibitory zone was against Candida albicans for its antifungal activity. Furthermore, AgNPs also showed a positive synergistic impact with the tested antibiotic and antifungal agents. In addition, the AgNPs demonstrated strong antioxidant activity. The considerable inhibition of alpha amylase and alpha glucosidase by the AgNPs were noted by their respective IC50 values of 16.65 μg/ml and 18.97 μg/ml. Flow cytometry analysis revealed a significant increase in glucose uptake by 3T3-L1 cells after exposure to AgNPs, as indicated by the upregulation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) expression. In 3T3/L cells, the AgNPs also demonstrated remarkable potential in accelerating wound healing achieving 92% closure of wounds after 48 h of incubation. As a result, it can be concluded that the AgNPs exhibit positive antidiabetic properties, suggesting their potential applications in the pharmaceutical industry including in the management of diabetic foot ulcers.

Construction of a Collagen-like Protein Based on Elastin-like Polypeptide Fusion and Evaluation of Its Performance in Promoting Wound Healing.

In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries.

Caspase-8 is required for HSV-1-induced apoptosis and promotes effective viral particle release via autophagy inhibition

Regulated cell death (RCD) plays an important role in the progression of viral replication and particle release in cells infected by herpes simplex virus-1 (HSV-1). However, the kind of RCD (apoptosis, necroptosis, others) and the resulting cytopathic effect of HSV-1 depends on the cell type and the species. In this study, we further investigated the molecular mechanisms of apoptosis induced by HSV-1. Although a role of caspase-8 has previously been suggested, we now clearly show that caspase-8 is required for HSV-1-induced apoptosis in a FADD-/death receptor-independent manner in both mouse embryo fibroblasts (MEF) and human monocytes (U937). While wild-type (wt) MEFs and U937 cells exhibited increased caspase-8 and caspase-3 activation and apoptosis after HSV-1 infection, respective caspase-8-deficient (caspase-8−/−) cells were largely impeded in any of these effects. Unexpectedly, caspase-8−/− MEF and U937 cells also showed less virus particle release associated with increased autophagy as evidenced by higher Beclin-1 and lower p62/SQSTM1 levels and increased LC3-I to LC3-II conversion. Confocal and electron microscopy revealed that HSV-1 stimulated a strong perinuclear multivesicular body response, resembling increased autophagy in caspase-8−/− cells, entrapping virions in cellular endosomes. Pharmacological inhibition of autophagy by wortmannin restored the ability of caspase-8−/− cells to release viral particles in similar amounts as in wt cells. Altogether our results support a non-canonical role of caspase-8 in both HSV-1-induced apoptosis and viral particle release through autophagic regulation.

Comparing Metabolomic and Essential Oil Fingerprints of Citrus australasica F. Muell (Finger Lime) Varieties and Their In Vitro Antioxidant Activity.

Comparative chemical analyses among peel and pulp essential oils (EOs) and methanolic extracts of four Citrus australasica varieties (Red, Collette, Pink Ice, and Yellow Sunshine), and the hybrid Faustrime, were performed using GC-MS and UHPLC-DAD-HR-Orbitrap/ESI-MS. Peel and pulp extracts were also analysed for their in vitro antioxidant activity on a Balb/3T3 clone A31 mouse embryo fibroblast cell line. The results of peel and pulp EOs were mainly characterised by monoterpenes and sesquiterpenes, respectively. All peels displayed a higher total phenol content (TPC) than pulps, and consequently a greater antioxidant activity. Collette peels and Pink Ice pulps showed the highest amount of identified flavonoids (e.g., luteolin, isosakuranetin, and poncirin derivatives). Collette and Red peels were rich in anthocyanins (delphinidin and petunidin glycosides), exhibiting the maximum protective activity against induced oxidative damage. In conclusion, finger lime fruits are good sources of health-promoting phytocomplexes, with the Red, Collette, and Pink Ice varieties being the most promising.

Characterization, Physical Properties, and Biocompatibility of Novel Tricalcium Silicate-Chitosan Endodontic Sealer.

The purpose of this study was to compare the characteristics, physical properties, and biocompatibility of the novel tricalcium silicate-chitosan (TCS-C) sealer with AH Plus and Sure-Seal Root. The TCS-C powder was prepared by mixing tricalcium silicate with 2% water-soluble chitosan at a 5:1 ratio, followed by sufficient addition of 10 g/mL ratio of double-distilled water to form a homogeneous cement. Material characterizations (the Fourier Transform InfraRed [FTIR] and X-ray diffraction [XRD]), physical property investigations (flow and film thickness), and cytotoxicity tests in 3T3 mouse embryo fibroblast cell (MTT assay method) were performed on sealers, and the results were compared with those of the commercial products. Statistical analysis was performed on flow and film thickness. The normality of the data was tested using the Shapiro-Wilk test. Statistical analysis was performed with one-way analysis of variance (ANOVA). The level of significance was set at p < 0.05. The TCS-C showed a mean flow of 31.98 ± 0.68 mm, compared with Sure Seal Root at 26.38 ± 0.69 mm and AH Plus at 26.50 ± 0.12 mm. The TCS-C showed a mean film thickness of 60 ± 10.0 mm compared with Sure-Seal Root at 50 ± 10.0 mm and AH Plus at 40 ± 15.8 mm. The TCS-C exhibited low to no cytotoxicity in fibroblast cell at all concentrations and exposure times. Adding water-soluble chitosan may improve the physical and biologic properties of tricalcium silicate cement. The novel TCS-C sealer did not fully meet the physical properties of an endodontic sealer, but it was not cytotoxic to fibroblast cells.

Youjing granules ameliorate spermatogenesis in rats through regulating the prolifereation of spermatogonial stem cells.

Male infertility has evolved from a common reproductive system disease to a major social issue. Youjing granule (YG) is a Chinese medicinal material used as a therapy method for tonifying the kidneys and removing dampness due to its pathogenic characteristics. YG has been shown to regulate sperm quality in clinical trials, but the underlying mechanism is not fully understood. The present study was aimed to explore the protective effects and mechanism of action of YG on male reproductive system damage caused by methyl methane sulfonate (MMS). We first established an infertility model of rats through oral administration of MMS and then treated with YG. To determine the effect of YG, spermatogenesis, microvascular density, and secretory function of Leydig cells and Sertoli cells in rats were assessed. Spermatogonial stem cells (SSCs) were co-cultured with mouse embryo fibroblast (MEF) cells as an in vitro cell model before exposure to serum containing YG. Furthermore, the proliferation and apoptosis of SSCs were measured. Results indicated that YG increased the expression of self-renewal and proliferation-related molecules such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2), and improved the quality of sperm and the proliferation of SSCs. In conclusion, YG may protect spermatogenetic function of rats through regulating the proliferation and self-renewal of SSCs.

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

A novel nanoparticle system targeting damaged mitochondria for the treatment of Parkinson's disease.

Mitochondrial damage is one of the primary causes of neuronal cell death in Parkinson's disease (PD). In PD patients, the mitochondrial damage can be repaired or irreversible. Therefore, mitochondrial damage repair becomes a promising strategy for PD treatment. In this research, hyaluronic acid nanoparticles (HA-NPs) of different molecular weights are used to protect the mitochondria and salvage the mild and limited damage in mitochondria. The HA-NPs with 2190k Dalton (kDa) HA can improve the mitochondrial function of SH-SY5Y cells and PTEN induced putative kinase 1 (PINK1) knockout mouse embryo fibroblast (MEF) cells. In cases of irreversible damage, NPs with ubiquitin specific peptidase 30 (USP30) siRNA are used to promote mitophagy. Meanwhile, by adding PINK1 antibodies, the NPs can selectively target the irreversibly damaged mitochondria, preventing the excessive clearance of healthy mitochondria.

Procollagen C-proteinase enhancer 1 promotes physiologic retinal angiogenesis via regulating the process of collagen.

To investigate the role of procollagen C-proteinase enhancer 1 (PCPE1) in retinal angiogenesis and relevant mechanisms. The Pcolce1-knockout (KO) mice were used to explore the effect of PCPE1 on retinal angiogenesis in vivo. Pcolce1 siRNA were designed, cell count kit 8 (CCK8) assays and tube formation assays were performed to investigate the cell proliferation and tube formation abilities of retinal microvascular endothelial cells (hRMECs) in vitro. Mouse embryo fibroblasts (MEF) cells were isolated and cultured to analyze the effect of PCPE1 on enhancing procollagen cleavage. In vivo studies showed that the retinal vascular density of Pcolce1-/- mice was significantly lower than that of the control group. Furthermore, silencing of Pcolce1 inhibited cell proliferation and tube formation abilities of hRMECs in vitro. Additionally, much more pro-collagen was found in Pcolce1-/- MEF cells, compared to wild type MEF cells. PCPE1 may promote physiological retinal angiogenesis by regulating the processing of collagen, which may provide a potential therapeutic target of retinal vascular disease.

Incorporation of Mycobacteriophage Fulbright into Polycaprolactone Electrospun Nanofiber Wound Dressing.

The Genus Mycobacterium includes pathogens known to cause disease in mammals such as tuberculosis (Mycobacterium tuberculosis) and skin infections (M. abscessus). M. smegmatis is a model bacterium that can cause opportunistic infections in human tissues and, rarely, a respiratory disease. Due to the emergence of multidrug-resistant bacteria, phage therapy is potentially an alternative way of treating these bacterial infections. As bacteriophages are specific to their bacterial host, it ensures that the normal flora is unharmed. Fulbright is a mycobacteriophage that infects the host bacteria M. smegmatis. The main goal of this study is to incorporate Mycobacteriophage Fulbright into a polycaprolactone (PCL) nanofiber and test its antimicrobial effect against the host bacteria, M. smegmatis. Stability tests conducted over 7 days showed that the phage titer does not decrease when in contact with PCL, making it a promising vehicle for phage delivery. Antimicrobial assays showed that PCL_Fulbright effectively reduces bacterial concentration after 24 h of contact. In addition, when stored at −20 °C, the phage remains viable for up to eleven months in the fiber. Fulbright addition on the nanofibrous mats resulted in an increase in water uptake and decrease in the mechanical properties (strength and Young’s modulus) of the membranes, indicating that the presence of phage Fulbright can greatly enhance the physical and mechanical properties of the PCL. Cytotoxicity assays showed that PCL_Fulbright is not cytotoxic to Balbc/3T3 mouse embryo fibroblast cell lines; thus, phage-incorporated PCL is a promising alternative to antibiotics in treating skin infections.

Development of Chitosan Silver Nanocomposites: Its Characteristic Study and Toxicity Effect against 3T3-L1 Cell Line

Nanocomposite from the natural source is opened a wide area for the researchers to find a natural remedy to replace the chemicals or harmful products in all the fields of agricultural, food and medical fields. Here the biopolymer (chitosan) was extracted from the two white rot fungi of Pleurotus floridanus and Pleurotus djamor, and biologically synthesized with 1mM AgNO3 solution. Synthesized chitosan nanocomposite was characterized with UV-Visible study, FTIR, FESEM, XRD, EDAS for the confirmation based upon the peaks, functional group, crystalline nature, size, morphology and the percentage of elements respectively. Toxicity study was carried out using 3T3 L1 (Mouse embryo fibroblast Cell Line) normal Cell Line to find out the cytotoxicity effect of the chitosan nanocomposite and found that the nanocomposites were non toxic to the Cell Line.

Photocatalytic performance and cytotoxic activity of green-synthesized cobalt ferrite nanoparticles

In this study, cobalt ferrite nanoparticles (CoFe2O4 NPs) were prepared using aqueous solution of torajabin followed by calcination at temperatures of 600, 700, and 800 °C. Synthesized nanoparticles of size 20–60 nm were characterized using PXRD, UV–Vis, FESEM, EDX, and Raman analysis. VSM analysis affirmed ferrimagnetic behavior. The photocatalytic degradation activity of CoFe2O4 NPs was assessed on Acid Orange 7 (AO7) dye under simulated sunlight exposure where high level of photodegradation was displayed by CF-600 NPs, due to their small particle size. The cytotoxic activity of CoFe2O4 NPs was studied on breast cancer cell (MCF7), colon cancer cell (CaCo2), and mouse embryo fibroblast cell (NIH-3T3) lines using MTT assay; nanoparticles were found to be nontoxic against cancer and normal cell lines. Thus, synthesized CoFe2O4 NPs could be an ideal choice for medical applications like drug delivery, and non-medical usages such as photodegradation of pollutants.

Novel N-(2-Methoxydibenzofuran-3-yl)-2-aryloxyacetamide Derivatives: Synthesis and Biological Investigation

Background: Dibenzofuran ring is a typical heterocyle that is found in many natural sources and its derivatives exhibit a wide scale of biological applications similar to its analog ring systems; furan and benzofuran. Materials and Methods: Novel N-(2-methoxydibenzofuran-3-yl)-2-aryloxyacetamide derivatives (2a-l) were synthesized and evaluated for their cytotoxic activity against A549 lung cancer and NIH/3T3 mouse embryofibroblast cell lines. The inhibition percentages of cathepsin D, L, acetylcholinesterase (AChE) and butrylcholinesterase (BuChE) enzymes provoked by the compounds were also determined. Results and Discussion: Most of the compounds exhibited significant cytotoxicity whose IC50 values were identified lower than the tested lowest concentration (&lt;3.90 μg/mL). Compound 2i against cathepsin D and compound 2k against cathepsin L displayed the highest inhibitory activity. Regrettably, the compounds demonstrated very weak AChE and BuChE inhibition. Conclusion: Compounds 2b, 2c, 2e, 2i and 2k exhibited the highest antiproliferative activity against A549 cell lines with selective profile. However, they did not display satisfying results on tested enzymes.

Abstract PR-003: Radiosensitization by targeting the NPM1/RAD51 axis

Abstract NPM1 is a chaperone involved in base excision, translesion synthesis and homologous recombination (HR). In the presence of DNA DSBs pT199 NPM1 co-localizes with H2AX foci, a consequence of binding to K63-linked ubiquitinated histones that surround DSBs via its pT199 domain comprised of an acidic tract and adjacent ubiquitin interacting motif-like domain (1-3). SUMOlyated K263 NPM1 binds to and promotes Rad51 loading onto resected 3-prime DNA at the DSB. Failure to SUMOlyate K263NPM or inhibition of NPM1 reduces Rad51 filament formation, thus inhibiting HR (4). A major effort is underway to develop DNA damage response inhibitors. The novel chemical entity YTR107 is a 5-((N-benzyl-1H-indol-3-yl) methylene) pyrimidine-2, 4,6(1H,3H,5H) trione developed using forward chemical genetics and structure/function screening (5). Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines (dose modifying factors of 1.5). Radiosensitization was NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line (3). Here we show that YTR107 inhibits radiation-induced NPM1 binding to Rad51, Rad 51 foci formation and repair of DNA DSBs.Tumor initiating cells (TICs) were radiosensitized by YTR107. YTR107 inhibited tumor growth (P = 0.027, 2-way ANOVA) and increased the survival of A549 tumor-bearing mice, determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107and 2.2 Gy(q.d. x 7) vs 20% for mice treated with 2.2 Gy alone (q.d.x 7). Cox Proportional log rank hazard ratio for irradiated vs unirradiated tumor-bearing mice was 0.24 (P = 0.0034) while the log rank hazard ratio for YTR107 plus irradiation vs unirradiated (± YTR107) tumor-bearing mice was 0.17 (P = 0.0023). Thus, these data provide support for development of YTR107 as a therapeutic radiation sensitizer. Supported in part by R44CA228756. 1.Koike A, Nishikawa H, Wu W, Okada Y, Venkitaraman AR, Ohta T.Cancer Res 2010;70:6746-56 2. Sekhar KR, Benamar M, Venkateswaran A, Sasi S, Penthala NR, Crooks PA, et al. Int J Radiat Oncol Biol Phys 2014;89:1106-14 3. Sekhar KR, Reddy YT, Reddy PN, Crooks PA, Venkateswaran A, McDonald WH, et al. Clin Cancer Res 2011;17:6490-9 4. Xu R, Yu S, Zhu D, Huang X, Xu Y, Lao Y, et al. Nat Commun 2019;10:3812 5. Reddy YT, Sekhar KR, Sasi N, Reddy PN, Freeman ML, Crooks PA. Bioorg Med Chem Lett 2010;20:600-2 Citation Format: Michael L. Freeman, Geri Traver, Konjeti R. Sekhar, Peter A. Crooks. Radiosensitization by targeting the NPM1/RAD51 axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-003.

The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals.

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

Inhibition of autophagy by 3‐methyladenine restricts murine cytomegalovirus replication

Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis.The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy.In cultured MEFs, the ratio of LC3I/II was reduced at 24 hourspost infection (hpi), but was increased at 48 hpiIn the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpiin MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups.Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.