Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein).

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP's influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands. KEY POINTS: • YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity. • YAP5SA regulates genes involved in proliferation, survival, and mTOR activation. • YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

PI3K-AKT/mTOR Signaling in Psychiatric Disorders: A Valuable Target to Stimulate or Suppress?

Economic development and increased stress have considerably increased the prevalence of psychiatric disorders in recent years, which rank as some of the most prevalent diseases globally. Several factors, including chronic social stress, genetic inheritance, and autogenous diseases, lead to the development and progression of psychiatric disorders. Clinical treatments for psychiatric disorders include psychotherapy, chemotherapy, and electric shock therapy. Although various achievements have been made researching psychiatric disorders, the pathogenesis of these diseases has not been fully understood yet, and serious adverse effects and resistance to antipsychotics are major obstacles to treating patients with psychiatric disorders. Recent studies have shown that the mammalian target of rapamycin (mTOR) is a central signaling hub that functions in nerve growth, synapse formation, and plasticity. The PI3K-AKT/mTOR pathway is a critical target for mediating the rapid antidepressant effects of these pharmacological agents in clinical and preclinical research. Abnormal PI3K-AKT/mTOR signaling is closely associated with the pathogenesis of several neurodevelopmental disorders. In this review, we focused on the role of mTOR signaling and the related aberrant neurogenesis in psychiatric disorders. Elucidating the neurobiology of the PI3K-AKT/mTOR signaling pathway in psychiatric disorders and its actions in response to antidepressants will help us better understand brain development and quickly identify new therapeutic targets for the treatment of these mental illnesses.

Alisol B 23-acetate promotes white adipose tissue browning to mitigate high-fat diet-induced obesity by regulating mTOR-SREBP1 signaling.

OBJECTIVE: Obesity is a global health concern with management strategies encompassing bariatric surgery and anti-obesity drugs; however, concerns regarding complexities and side effects persist, driving research for more effective, low-risk strategies. The promotion of white adipose tissue (WAT) browning has emerged as a promising approach. Moreover, alisol B 23-acetate (AB23A) has demonstrated efficacy in addressing metabolic disorders, suggesting its potential as a therapeutic agent in obesity management. Therefore, in this study, we aimed to investigate the therapeutic potential of AB23A for mitigating obesity by regulating metabolic phenotypes and lipid distribution in mice fed a high-fat diet (HFD). METHODS: An obesity mouse model was established by administration of an HFD. Glucose and insulin metabolism were assessed via glucose and insulin tolerance tests. Adipocyte size was determined using hematoxylin and eosin staining. The expression of browning markers in WAT was evaluated using Western blotting and quantitative real-time polymerase chain reaction. Metabolic cage monitoring involved the assessment of various parameters, including food and water intake, energy metabolism, respiratory exchange rates, and physical activity. Moreover, oil red O staining was used to evaluate intracellular lipid accumulation. A bioinformatic analysis tool for identifying the molecular mechanisms of traditional Chinese medicine was used to examine AB23A targets and associated signaling pathways. RESULTS: AB23A administration significantly reduced the weight of obese mice, decreased the mass of inguinal WAT, epididymal WAT, and perirenal adipose tissue, improved glucose and insulin metabolism, and reduced adipocyte size. Moreover, treatment with AB23A promoted the expression of browning markers in WAT, enhanced overall energy metabolism in mice, and had no discernible effect on food intake, water consumption, or physical activity. In 3T3-L1 cells, AB23A inhibited lipid accumulation, and both AB23A and rapamycin inhibited the mammalian target of rapamycin-sterol regulatory element-binding protein-1 (mTOR-SREBP1) signaling pathway. Furthermore, 3-isobutyl-1-methylxanthine, dexamethasone and insulin, at concentrations of 0.25 mmol/L, 0.25 µmol/L and 1 µg/mL, respectively, induced activation of the mTOR-SREBP1 signaling pathway, which was further strengthened by an mTOR activator MHY1485. Notably, MHY1485 reversed the beneficial effects of AB23A in 3T3-L1 cells. CONCLUSION: AB23A promoted WAT browning by inhibiting the mTOR-SREBP1 signaling pathway, offering a potential strategy to prevent obesity. Please cite this article as: Han LL, Zhang X, Zhang H, Li T, Zhao YC, Tian MH, Sun FL, Feng B. Alisol B 23-acetate promotes white adipose tissue browning to mitigate high-fat diet-induced obesity by regulating mTOR-SREBP1 signaling. J Integr Med. 2024; 22(1): 83-92.

Temsirolimus (Torisel®) en tumor desmoplásico de células redondas avanzado / Temsirolimus (Torisel®) in desmoplastic tumor of advanced round cells

INTRODUCCIÓN: El tumor desmoplásico de células redondas es un tumor mesenquimal que surge de la superficie peritoneal. 1 Es considerado un sarcoma de partes blandas que suele afectar principalmente al sexo masculino con una elevada prevalencia en niños y adultos jóvenes. 1,2 En Argentina se desconoce su incidencia, pero es considerado una neoplasia poco frecuente. La tasa de incidencia ajustada por edad en los Estados Unidos se estima en 0,3 casos/millón de habitantes.3 Su principal característica diagnóstica es una traslocación recíproca del gen t(11;22) (p13;q11 o q12), que provoca la fusión del gen del sarcoma de Ewing y el cromosoma 22 del tumor de Wilms. 1 Las manifestaciones clínicas iniciales suelen ser inespecíficas y están relacionadas con el tamaño, la ubicación y la velocidad de progresión de la enfermedad.1,2 Suele presentarse como una masa abdominal palpable con dolor y otros síntomas gastrointestinales asociados. Las masas tumorales pueden causar síntomas de compresión, como obstrucción intestinal y uronefrosis. En casos avanzados puede existir carcinomatosis peritoneal con o sin ascitis, o compromiso metastásico en ganglios linfáticos, hígado y pulmones. TECNOLOGÍA: El temsirolimus es un inhibidor selectivo de la molécula denominada mTOR (diana de la rapamicina en las células de mamífero).6,7 Además de regular las proteínas del ciclo celular, mTOR regula la traducción de los factores inducibles por la hipoxia, HIF-1 y HIF-2 alfa. Estos factores regulan la capacidad de los tumores de adaptarse a entornos hipóxicos y de producir el factor de crecimiento del endotelio vascular (VEGF). Por tanto, el efecto antitumoral del temsirolimus podría derivar también en parte de su capacidad de deprimir los niveles de HIF y VEGF en el tumor o en el microentorno tumoral y reducir así el desarrollo de vasos. OBJETIVO: El objetivo del presente informe es evaluar rápidamente los parámetros de eficacia, seguridad, costos y recomendaciones disponibles acerca del empleo de temsirolimus (Torisel®) en personas con diagnóstico de tumor desmoplásico de células redondas avanzado. MÉTODOS: Se realizó una búsqueda bibliográfica en las principales bases de datos tales como PUBMED, LILACS, BRISA, COCHRANE, SCIELO, EMBASE, TRIPDATABASE como así también en sociedades científicas, agencias reguladoras, financiadores de salud y agencias de evaluación de tecnologías sanitarias. Se priorizó la inclusión de revisiones sistemáticas, ensayos clínicos controlados aleatorizados, evaluación de tecnología sanitaria y guías de práctica clínica de alta calidad metodológica. RECOMENDACIONES No se hallaron guías de práctica clínica actualizadas en Argentina y en el Mundo que recomienden la tecnología en la indicación evaluada. Las guías actualizadas de la Red Nacional de Centros para el Tratamiento Integral del cáncer de los Estados Unidos (NCCN, su sigla del inglés National Comprehensive Cáncer Network) no mencionan el uso de temsirolimus dentro de las opciones terapéuticas para el tratamiento de los tumores desmoplásicos de células redondas avanzados.4 Un consenso de expertos estadounidenses sobre la temática publicado en 2020 no menciona el uso de temsirolimus dentro de las opciones de tratamento. CONCLUSIONES No se hallaron ensayos clínicos publicados que hayan evaluado la eficacia y seguridad de la utilización de temsirolimus (Torisel®) en personas con diagnóstico de tumor desmoplásico de células redondas avanzado. La evidencia que sustentaría el uso del fármaco en la indicación mencionada proviene de una series de casos retrospectiva. El mismo incluyo cinco personas progresadas a distintos tratamientos y cirugías, que utilizaron temsirolimus asociada a vinorelbina y ciclofosfamida. El estudio reportó una mediana de libre de progresión de 8,5 meses sobre tres personas, y eventos adversos serios como la fatiga, neutropenia grado 3-4, mucositis y fallo renal. Un estudio en curso, sin resultados publicados y que incluye la tecnología en la indicación evaluada, ha detenido la inclusión de personas debido a que no se cumplió su objetivo de eficacia pre-especificado. Su comercialización en la indicación evaluada no se encuentra autorizada por la agencia regulatoria de los Estados Unidos y de Europa. No se hallaron guías de práctica clínica actualizadas en Argentina y en el Mundo que recomienden la tecnología en la indicación evaluada. No se hallaron evaluaciones económicas publicadas para Argentina y el Mundo, donde el precio de adquisición internacional de referencia relevado para un vial de 25mg/ml es de USD 1.213 (ARS 443.352 noviembre/23).

Assessment of the anti-inflammatory mechanism of quercetin 3,7-dirhamnoside using an integrated pharmacology strategy.

Pouzolzia zeylanica (L.) Benn. is a Chinese herbal medicine widely used for its anti-inflammatory and pus-removal properties. To explore its potential anti-inflammatory mechanism, quercetin 3,7-dirhamnoside (QDR), the main flavonoid component of P. zeylanica (L.) Benn., was extracted and purified. The potential anti-inflammatory targets of QDR were predicted using network analysis. These potential targets were verified using molecular docking, molecular dynamics simulations, and in vitro experiments. Consequently, 342 potential anti-inflammatory QDR targets were identified. By analyzing the intersection between the protein-protein interaction and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified several potential protein targets of QDR, including RAC-alpha serine/threonine-protein kinase (AKT1), Ras-related C3 botulinum toxin substrate 1 (RAC1), nitric oxide synthase 3 (NOS3), serine/threonine-protein kinase mTOR (mTOR), epidermal growth factor receptor (EGFR), growth factor receptor-bound protein 2 (GRB2), and endothelin-1 receptor (EDNRA). QDR has anti-inflammatory activity and regulates immune responses and apoptosis through chemokines, Phosphatidylinositol 3-kinase 3(PI3K)/AKT, cAMP, T-cell receptor, and Ras signaling pathways. Molecular docking analysis showed that QDR has good binding abilities with AKT1, mTOR, and NOS3. In addition, molecular dynamics simulations demonstrated that the protein-ligand complex systems formed between QDR and AKT1, mTOR, and NOS3 have high dynamic stability, and their protein-ligand complex systems possess strong binding ability. In RAW264.7 macrophages, QDR significantly inhibited lipopolysaccharides (LPS)-induced inducible nitric oxide synthase expression, nitric oxide (NO) release and the generation of proinflammatory cytokines IL-6, IL-1ß, and TNF-α. QDR downregulated the expression of p-AKT1(Ser473)/AKT1 and p-mTOR (Ser2448)/mTOR, and upregulated the expression of NOS3, Rictor, and Raptor. This indicates that the anti-inflammatory mechanisms of QDR involve regulation of AKT1 and mTOR to prevent apoptosis and of NOS3 which leads to the release of endothelial NO. Thus, our study elucidated the potential anti-inflammatory mechanism of QDR, the main flavonoid found in P. zeylanica (L.) Benn.

Nephrotoxicity assessment of podophyllotoxin-induced rats by regulating PI3K/Akt/mTOR-Nrf2/HO1 pathway in view of toxicological evidence chain (TEC) concept.

Adverse reactions to traditional Chinese medicine have hindered the healthy development and internationalization process of the traditional Chinese medicine industry. The critical issue that needs to be solved urgently is to evaluate the safety of traditional Chinese medicine systematically and effectively. Podophyllotoxin (PPT) is a highly active compound extracted from plants of the genus Podophyllum such as Dysosma versipellis (DV). However, its high toxicity and toxicity to multiple target organs affect the clinical application, such as the liver and kidney. Based on the concurrent effects of PPT's medicinal activity and toxicity, it would be a good example to conduct a systematic review of its safety. Therefore, this study revolves around the Toxicological Evidence Chain (TEC) concept. Based on PPT as the main toxic constituent in DV, observe the objective toxicity impairment phenotype of animals. Evaluate the serum biochemical indicators and pathological tissue sections for substantial toxic damage results. Using metabolomics, lipidomics, and network toxicology to evaluate the nephrotoxicity of PPT from multiple perspectives systematically. The results showed that PPT-induced nephrotoxicity manifested as renal tubular damage, mainly affecting metabolic pathways such as glycerophospholipid metabolism and sphingolipid metabolism. PPT inhibits the autophagy process of kidney cells through the PI3K/Akt/mTOR and Nrf2/HO1 pathways and induces the activation of oxidative stress in the body, thereby causing nephrotoxic injury. This study fully verified the feasibility of the TEC concept for the safety and toxicity evaluation of traditional Chinese medicine. Provide a research template for systematically evaluating the safety of traditional Chinese medicine.

Statins in Kidney Transplant Recipients: Usage, All-Cause Mortality, and Interactions with Maintenance Immunosuppressive Agents.

SIGNIFICANCE STATEMENT: Cardiovascular diseases account for 32% of deaths among kidney transplant recipients. Statin therapy is common in this population. However, its effect on mortality prevention remains unclear among kidney transplant recipients, whose clinical risk profile might be unique because of concomitant immunosuppressive therapy. In this national study of 58,264 single-kidney transplant recipients, statin use was associated with a 5% decrease in mortality. More importantly, this protective association was stronger among those who used a mammalian target of rapamycin (mTOR) inhibitor for immunosuppression (27% decrease in mTOR inhibitor users versus 5% in nonusers). Our results suggest that statin therapy may reduce mortality in kidney transplant recipients and that the strength of this protective association may vary by immunosuppression regimen. BACKGROUND: Cardiovascular diseases are the leading cause of mortality in kidney transplant (KT) recipients, accounting for 32% of deaths. Statins are widely used in KT recipients, but effectiveness for preventing mortality remains unclear in this population, especially because of interaction between statins and immunosuppressive agents. We analyzed a national cohort to assess the real-world effectiveness of statins for reducing all-cause mortality in KT recipients. METHODS: We studied statin use and mortality among 58,264 adults (18 years or older) who received single kidneys between 2006 and 2016 and had Medicare part A/B/D. Statin use was ascertained from Medicare prescription drug claims and deaths from Center for Medicare and Medicaid Services records. We estimated the association of statin use with mortality using multivariable Cox models, with statin use as a time-varying exposure and immunosuppression regimen as effect modifiers. RESULTS: Statin use increased from 45.5% at KT to 58.2% at 1-year post-KT to 70.9% at 5-year post-KT. We observed 9785 deaths over 236,944 person-years. Overall, statin use was significantly associated with lower mortality (adjusted hazard ratio [aHR], 0.95; 95% confidence interval [CI], 0.90 to 0.99). The strength of this protective association varied by calcineurin inhibitor use (among tacrolimus users, aHR, 0.97; 95% CI, 0.92 to 1.03 versus among calcineurin nonusers, aHR, 0.72; 95% CI, 0.60 to 0.87; interaction P =0.002), mammalian target of rapamycin (mTOR) inhibitor use (among mTOR inhibitor users, aHR, 0.73; 95% CI, 0.57 to 0.92 versus among nonusers, aHR, 0.95; 95% CI, 0.91 to 1.00; interaction P =0.03), and mycophenolate use (among mycophenolate users, aHR, 0.96; 95% CI, 0.91 to 1.02 versus among nonusers, aHR, 0.76; 95% CI, 0.64 to 0.89; interaction P =0.002). CONCLUSION: Real-world evidence supports statin therapy for reducing all-cause mortality in KT recipients. Effectiveness might be greater when combined with mTOR inhibitor-based immunosuppression.

SARS-CoV-2 vaccine alleviates disease burden and severity in liver transplant recipients even with low antibody titers.

BACKGROUND AND AIMS: We retrospectively assessed the clinical Pfizer's mRNA SARS-CoV-2 BNT162b2 vaccination outcomes and the serologic impact on liver transplant (LT) recipients. PATIENTS AND METHODS: One hundred and sixty-seven LT cases followed between March 1, 2020 and September 25, 2021, and were stratified into two groups: (1) 37 LT recipients after SARS-CoV-2 infection before vaccine era and (2) 130 LT recipients vaccinated with 2 doses without earlier SARS-CoV-2 exposure. Serum SARS-CoV-2 spike immunoglobulins (anti-S) were assessed 7 days following vaccination (Liaison assay). RESULTS: In addition to the 37 nonvaccinated cases (22.2% of total group) who experienced SARS-CoV-2 infection (34 symptomatic and 3 asymptomatic), another 8 vaccinated symptomatic recipients (4.8%) were infected (5 from the third and three from the fourth waves). Three of the 45 infected cases died (6.7%) before the vaccine program. Vaccinated group: of the 130 LT vaccinated recipients, 8 (6.2%) got infected postvaccination (added to the infected group) and were defined as clinical vaccine failure; 38 (29.2%) were serological vaccine failure (total failure 35.4%), and 64.6% cases were serological vaccine responders (anti-S≥19 AU/mL). Longer post-LT interval and lower consumption of immunosuppressants (steroids, FK506, and mycophenolate mofetil) correlated with favorable SARS-CoV-2 vaccine response. Mammalian target of rapamycin inhibitors improved vaccine outcomes associated with lower FK506 dosages and serum levels. Patients with anti-S levels <100 AU/mL risked losing serologic response or being infected with SARS-CoV-2. A booster dose achieved an effective serologic response in a third of failures and most responders, securing better and possibly longer protection. CONCLUSION: Pfizer's BNT162b2 vaccine seems to lessen SARS-CoV-2 morbidity and mortality of LT recipients even with weak serological immunogenicity. Switching mycophenolate mofetil to mammalian target of rapamycin inhibitors might be effective before boosters in vaccine failure cases. A booster vaccine should be considered for nonresponders and low-responders after the second dose.

Disease monitoring using lung function trajectory in lymphangioleiomyomatosis: assessment in two national cohorts.

STUDY QUESTION: In lymphangioleiomyomatosis, airflow obstruction and impairment of gas transfer progress at variable rates and serial lung function is recommended for disease monitoring. As these measurements are variable, recognising subjects needing treatment can be difficult. We used two prospective national cohorts to study change over time and variation in FEV1 to inform clinical decision making. PATIENTS AND METHODS: Clinical and lung function data for 141 UK and 148 American subjects were studied. Multilevel mixed effects modelling, route mean square analysis of errors and Bland-Altman analysis were used to analyse variability in lung function over time. RESULTS: At baseline assessment, DLCO was reduced to a greater degree than FEV1. In untreated patients, FEV1 and DLCO declined at proportionately similar rates independent of initial lung function. In mechanistic target of rapamycin (mTOR) inhibitor treated patients, FEV1 stabilised but DLCO continued to decline. FEV1/DLCO per cent predicted ratio was 1.37 (0.43) at baseline and increased to 1.41 (0.50) after 42 (24) months (p=0.0002). At least five measurements were required before >70% of individuals had estimates of rate of FEV1 loss within 50 mL/year and DLCO loss within 0.1 mmol/min/kPa/year of the final values. CONCLUSIONS: While FEV1 and DLCO fall proportionately in most, in early disease and during mTOR inhibitor treatment, DLCO should also be monitored as it may fall independent of FEV1. Since at least five observations over many months are required to make confident estimates of FEV1 and DLCO trajectories, new strategies are needed to measure disease activity and target early treatment appropriately.

Use of mTOR inhibitors (rapalogs) for the treatment of skin changes in tuberous sclerosis complex.

BACKGROUND: Skin manifestations of Tuberous Sclerosis Complex (TSC) are present in more than 90% of patients. Facial angiofibromas (AF) are considered a skin hallmark of TSC. They are responsible for esthetic impact in patients. We aimed at reviewing the data available on the use of rapamycin (sirolimus) and everolimus for the oral or topical treatment of AF and other TSC-related skin changes and reporting our preliminary experience at Angers University Hospital. METHODS: The literature search has been performed in combining the terms "rapamycin", "sirolimus", "everolimus", "tuberous sclerosis complex", "skin" and "trial". We have splited the findings of the literature search into two parts: 1) the value of rapalogs used systemically for extracutaneous purposes and 2) the role of topical rapalogs used specifically for skin lesions. RESULTS: Large clinical trials using rapamycin or everolimus for the treatment of brain, lung or kidney manifestations of TSC unfortunately poorly define the "skin lesion response rate" they report. Conversely, the trials with topical rapamycin demonstrate significant, albeit transient, efficacy on AF size and visibility and acceptable tolerance. Several trials suggest better efficacy in younger patients than in adults. Long-term evaluation (up to 136 weeks) point to sustained response and good local and systemic tolerance. However, maintenance therapy appears to be mandatory to preserve skin response. Other skin changes, especially shagreen fibrotic plaques, hypomelanotic macules and ungual tumors still need far more research. Our experience in 124 patients (children and adults) treated for facial AF at Angers University Hospital showed that about 80% of them had an impressive and sustained response. CONCLUSION: The issues of cost and access to affordable topical rapamycin formulations are critical for the patients even if skin changes do not cause serious harm in the context of TSC. We strongly suggest to improve and standardize the formulation of topical rapamycin, to encourage the pharmaceutical industry for providing commercial products, and the Health systems (social welfare) to reimburse them. © 2022 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Quantifying the Value of the Molecular Tumor Board: Discordance Recommendation Rate and Drug Cost Avoidance.

PURPOSE: Molecular tumor boards (MTBs) provide interventions that assist the patient's primary oncologist's interpretation and application of precision oncology and avoid clinical and financial toxicities of prescribing inappropriate targeted therapy. In this article, we describe a novel method for illustrating MTBs value and recommendation discordance rate and report associated drug cost avoidance data. METHODS: From January 1, 2021, to December 31, 2021, patients assessed by our program's MTB were retrospectively evaluated. Recommendation discordance was defined as any disagreement between MTB therapeutic recommendations and those provided in the next-generation sequencing vendor's report. RESULTS: In 2021, our program processed 1,119 next-generation sequencing orders via external vendors for 1,029 unique patients with a variety of solid tumor and hematologic malignancies. During this period, 962 patients were reviewed through our MTB process. MTB recommendation discordance rate was high (229 of 502; 45.6%) and varied across test vendors. Rationales for discordance included the following: low level of evidence (88% of patients), alternative standard of care available (60%), and tolerability concerns (42%), among others. Discordance was highest for Vendor C (30%), followed by Vendor A (24%) and Vendor B (8%). The most common drug classes not supported were mTOR, PARP, MEK, and PIK3CA inhibitors when recommended by vendors in off-label settings. MTB interventions accounted for $3,209,070 in US dollars in potential drug cost avoidance. CONCLUSION: Therapeutic recommendation discordance rates can provide quantitative insight into the benefit of MTB. Discordance-associated drug cost avoidance further demonstrates MTB's financial value. These measures may be used as part of the justification for this service line within a cancer care program.

Investigation of cytotoxic and apoptotic effects of disodium pentaborate decahydrate on ovarian cancer cells and assessment of gene profiling.

After revealing the anti-cancer properties of boron, which is included in the category of essential elements for human health by the World Health Organization, the therapeutic potential of boron compounds has been begun to be evaluated, and its molecular effect mechanisms have still been among the research subjects. In ovarian cancer, mutations or amplifications frequently occur in the PI3K/Akt/mTOR pathway components, and dysregulation of this pathway is shown among the causes of treatment failure. In the present study, it was aimed to investigate the anti-cancer properties of boron-containing DPD in SKOV3 cells, which is an epithelial ovarian cancer model, through PI3K/AKT/mTOR pathway. The cytotoxic activity of DPD in SKOV3 cells was evaluated by WST-1 test, apoptotic effect by Annexin V and JC-1 test. The gene expressions associated with PI3K/AKT/mTOR pathway were determined by real-time qRT-PCR. In SKOV3 cells, the IC50 value of DPD was found to be 6.7 mM, 5.6 mM, and 5.2 mM at 24th, 48th and 72nd hour, respectively. Compared with the untreated control group, DPD treatment was found to induce apoptosis 2.6-fold and increase mitochondrial membrane depolarization 4.5-fold. DPD treatment was found to downregulate PIK3CA, PIK3CG, AKT2, IGF1, IRS1, MAPK3, HIF-1, VEGFC, CAB39, CAB39L, STRADB, PRKAB2, PRKAG3, TELO2, RICTOR, MLST8, and EIF4B genes and upregulate TP53, GSK3B, FKBP8, TSC2, ULK1, and ULK2 genes. These results draw attention to the therapeutic potential of DPD, which is frequently exposed in daily life, in epithelial ovarian cancer and show that it can be a candidate compound in combination with chemotherapeutics.

Immunoblotting-assisted assessment of JAK/STAT and PI3K/Akt/mTOR signaling in myeloproliferative neoplasms CD34+ stem cells.

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.

Mammalian Target of Rapamycin Pathway Assessment in Antiphospholipid Antibody-Positive Patients with Livedo.

OBJECTIVE: In antiphospholipid antibody (aPL) nephropathy, activation of the mammalian target of rapamycin (mTOR) contributes to endothelial cell proliferation, a key finding of aPL microvascular disease. Here, we examined mTOR activation in the skin of aPL-positive patients with livedo. METHODS: Three patient groups with livedo were studied: (1) persistently aPL-positive with systemic lupus erythematosus (SLE); (2) persistently aPL-positive without SLE; and (3) aPL-negative SLE (control). After collecting aPL-related medical history, two 5-mm skin biopsies of livedo were performed on each patient: (1) peripheral (erythematous-violaceous lesion); and (2) central (nonviolaceous area). We stained specimens for phosphorylated protein kinase B (p-AKT) and phosphorylated S6 ribosomal protein (p-S6RP) as mTOR activity markers, CD31 to identify endothelial cells, and Ki-67 to show cellular proliferation. We counted cells in the epidermis and compared mTOR-positive cell counts between peripheral and central samples, and between patient groups, using Freidman test and Wilcoxon signed-rank test. RESULTS: Ten patients with livedo reticularis were enrolled: 4 aPL-positive without SLE (antiphospholipid syndrome [APS] classification met, n = 3), 4 aPL-positive SLE (APS classification met, n = 3), and 2 aPL-negative SLE (control). In all aPL-positive patients, epidermal p-AKT and p-S6RP staining were significantly increased in both peripheral and central skin samples when compared to aPL-negative SLE controls; both were more pronounced in the lower basal layers of epidermis. CONCLUSION: Our study demonstrates increased mTOR activity in livedoid lesions of aPL-positive patients with or without SLE compared to aPL-negative patients with SLE, with more prominent activity in the lower basal layers of the epidermis. These findings may serve as a basis for further investigating the mTOR pathway in aPL-positive patients.

Kappa-Opioid Receptor Blockade Ameliorates Obesity Caused by Estrogen Withdrawal via Promotion of Energy Expenditure through mTOR Pathway.

Weight gain is a hallmark of decreased estradiol (E2) levels because of menopause or following surgical ovariectomy (OVX) at younger ages. Of note, this weight gain tends to be around the abdomen, which is frequently associated with impaired metabolic homeostasis and greater cardiovascular risk in both rodents and humans. However, the molecular underpinnings and the neuronal basis for these effects remain to be elucidated. The aim of this study is to elucidate whether the kappa-opioid receptor (k-OR) system is involved in mediating body weight changes associated with E2 withdrawal. Here, we document that body weight gain induced by OVX occurs, at least partially, in a k-OR dependent manner, by modulation of energy expenditure independently of food intake as assessed in Oprk1-/-global KO mice. These effects were also observed following central pharmacological blockade of the k-OR system using the k-OR-selective antagonist PF-04455242 in wild type mice, in which we also observed a decrease in OVX-induced weight gain associated with increased UCP1 positive immunostaining in brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Remarkably, the hypothalamic mTOR pathway plays an important role in regulating weight gain and adiposity in OVX mice. These findings will help to define new therapies to manage metabolic disorders associated with low/null E2 levels based on the modulation of central k-OR signaling.

Assessment of mitophagy in human iPSC-derived cardiomyocytes.

Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.Abbreviations: AAVS1: adeno-associated virus integration site 1; AKT/protein kinase B: AKT serine/threonine kinase; CAG promoter: cytomegalovirus early enhancer, chicken ACTB/ß-actin promoter; CIS: cisplatin; CRISPR: clustered regularly interspaced short palindromic repeats; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; hiPSC: human induced pluripotent stem cell; hiPSC-CMs: human induced pluripotent stem cell-derived cardiomyocytes; ISO: isoproterenol; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RT: room temperature; SB: SBI-0206965; ULK1: unc-51 like autophagy activating kinase 1.

Experimental and computational assessment of the synergistic pharmacodynamic drug-drug interactions of a triple combination therapy in refractory HER2-positive breast cancer cells.

The development of innate and/or acquired resistance to human epidermal growth factor receptor type-2 (HER2)-targeted therapy in HER2-positive breast cancer (HER2 + BC) is a major clinical challenge that needs to be addressed. One of the main mechanisms of resistance includes aberrant activation of the HER2 and phosphatidylinositol 3-kinase/AKT8 virus oncogene cellular homolog/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways. In the present work, we propose to use a triple combination therapy to combat this resistance phenomenon. Our strategy involves evaluation of two targeted small molecule agents, everolimus and dasatinib, with complementary inhibitory circuitries in the PI3K/Akt/mTOR pathway, along with a standard cytotoxic agent, paclitaxel. Everolimus inhibits mTOR, while dasatinib inhibits Src, which is a protein upstream of Akt. An over-activation of these two proteins has been implicated in approximately 50% of HER2 + BC cases. Hence, we hypothesize that their simultaneous inhibition may lead to enhanced cell-growth inhibition. Moreover, the potent apoptotic effects of paclitaxel may help augment the overall cytotoxicity of the proposed triple combination in HER2 + BC cells. To this end, we investigated experimentally and assessed computationally the in vitro pharmacodynamic drug-drug interactions of the various dual and triple combinations to assess their subsequent combinatorial effects (synergistic/additive/antagonistic) in a HER2-therapy resistant BC cell line, JIMT-1. Our proposed triple combination therapy demonstrated synergism in JIMT-1 cells, thus corroborating our hypothesis. This effort may form the basis for further investigation of the triple combination therapy in vivo at a mechanistic level in HER2-therapy resistant BC cells.

Protocol for the assessment of mTOR activity in mouse primary hepatocytes.

We present a protocol for measuring the activity of the mechanistic target of rapamycin (mTOR) pathway in ex vivo isolated mouse primary hepatocytes. It can be used as a tool for genetic, pharmacological, metabolomic, and signal transduction procedures. We discuss critical aspects for improving yield, viability, and modulation of the mTOR pathway. This protocol can be adapted to other signaling cascades and is compatible with multiple readouts. For complete details on the use and execution of this protocol, please refer to Ortega-Molina et al. (2021).

Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation.

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2-3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

Assessment of AXL and mTOR genes expression in medullary thyroid carcinoma (MTC) cell line in relation with over expression of miR-144 and miR-34a.

OBJECTIVES: The aim of the present study was to investigate the expression of AXL and mTOR genes and their targeting microRNAs (miRNAs) including miR-34a and miR-144 in Medullary Thyroid Carcinoma (MTC) cell line, TT, and determine the effect of these two miRNAs on their target genes to introduce new molecular markers or therapeutics. METHODS: The expression of miR-34a, miR-144, and their targets genes including AXL and mTOR was evaluated by quantitative Real-time PCR. Luciferase assay was performed to confirm the interaction between miRNAs and their target mRNAs. The expression level of AXL and mTOR was evaluated before and after miRNAs induction in TT cell line compared with Cos7 as control cells. RESULTS: The expression of AXL and mTOR were up-regulated significantly, while miR-34a and miR-144 were down-regulated in TT cell line compared to Cos7. After transduction, the overexpression of miR-34a and 144 caused down-regulation of both genes. Luciferase assay results showed that the mTOR is targeted by miR-34a and miR-144 and the intensity of luciferase decreased in the presence of miRNAs. CONCLUSIONS: Based on the results of the present study and since AXL and mTOR genes play a critical role in variety of human cancers, suppression of these genes by their targeting miRNAs, especially miR-34a and miR-144, can be propose as a new strategy for MTC management. However, more studies are needed to approve the hypothesis.