Harnessing the Electrochemical Effects of Electroporation-Based Therapies to Enhance Anti-tumor Immune Responses.

This study introduces a new method of targeting acidosis (low pH) within the tumor microenvironment (TME) through the use of cathodic electrochemical reactions (CER). Low pH is oncogenic by supporting immunosuppression. Electrochemical reactions create local pH effects when a current passes through an electrolytic substrate such as biological tissue. Electrolysis has been used with electroporation (destabilization of the lipid bilayer via an applied electric potential) to increase cell death areas. However, the regulated increase of pH through only the cathode electrode has been ignored as a possible method to alleviate TME acidosis, which could provide substantial immunotherapeutic benefits. Here, we show through ex vivo modeling that CERs can intentionally elevate pH to an anti-tumor level and that increased alkalinity promotes activation of naïve macrophages. This study shows the potential of CERs to improve acidity within the TME and that it has the potential to be paired with existing electric field-based cancer therapies or as a stand-alone therapy.

Extracellular matrix detached cancer cells resist oxidative stress by increasing histone demethylase KDM6 activity.

Epithelial cancer cells rely on the extracellular matrix (ECM) attachment in order to spread to other organs. Detachment from the ECM is necessary for these cells to seed in other locations. When the attachment to the ECM is lost, cellular metabolism undergoes a significant shift from oxidative metabolism to glycolysis. Additionally, the cancer cells become more dependent on glutaminolysis to avoid a specific type of cell death known as anoikis, which is associated with ECM detachment. In our recent study, we observed increased expression of H3K27me3 demethylases, specifically KDM6A/B, in cancer cells that were resistant to anoikis. Since KDM6A/B is known to regulate cellular metabolism, we investigated the effects of suppressing KDM6A/B with GSK-J4 on the metabolic processes in these anoikis-resistant cancer cells. Our results from untargeted metabolomics revealed a profound impact of KDM6A/B inhibition on various metabolic pathways, including glycolysis, methyl histidine, spermine, and glutamate metabolism. Inhibition of KDM6A/B led to elevated reactive oxygen species (ROS) levels and depolarization of mitochondria, while reducing the levels of glutathione, an important antioxidant, by diminishing the intermediates of the glutamate pathway. Glutamate is crucial for maintaining a pool of reduced glutathione. Furthermore, we discovered that KDM6A/B regulates the key glycolytic genes expression like hexokinase, lactate dehydrogenase, and GLUT-1, which are essential for sustaining glycolysis in anoikis-resistant cancer cells. Overall, our findings demonstrated the critical role of KDM6A/B in maintaining glycolysis, glutamate metabolism, and glutathione levels. Inhibition of KDM6A/B disrupts these metabolic processes, leading to increased ROS levels and triggering cell death in anoikis-resistant cancer cells.

Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model.

Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.

Global epidemiology of breast cancer based on risk factors: a systematic review.

Background: Numerous reviews of the epidemiology and risk factors for breast cancer have been published previously which heighted different directions of breast cancer. Aim: The present review examined the likelihood that incidence, prevalence, and particular risk factors might vary by geographic region and possibly by food and cultural practices as well. Methods: A systematic review (2017-2022) was conducted following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, reporting on epidemiological and risk factor reports from different world regions. Medical Subject Heading (MeSH) terms: "Breast neoplasm" "AND" country terms such as "Pakistan/epidemiology", "India/epidemiology", "North America/epidemiology", "South Africa/epidemiology" were used to retrieve 2068 articles from PubMed. After applying inclusion and exclusion terms, 49 papers were selected for systematic review. Results: Results of selected articles were summarized based on risk factors, world regions and study type. Risk factors were classified into five categories: demographic, genetic and lifestyle risk factors varied among countries. This review article covers a variety of topics, including regions, main findings, and associated risk factors such as genetic factors, and lifestyle. Several studies revealed that lifestyle choices including diet and exercise could affect a person's chance of developing breast cancer. Breast cancer risk has also been linked to genetic variables, including DNA repair gene polymorphisms and mutations in the breast cancer gene (BRCA). It has been found that most of the genetic variability links to the population of Asia while the cause of breast cancer due to lifestyle modifications has been found in American and British people, indicating that demographic, genetic, and, lifestyle risk factors varied among countries. Conclusion: There are many risk factors for breast cancer, which vary in their importance depending on the world region. However, further investigation is required to better comprehend the particular causes of breast cancer in these areas as well as to create efficient prevention and treatment plans that cater to the local population.

Successful In Situ Targeting of Pancreatic Tumors in a Novel Orthotopic Porcine Model Using Histotripsy.

OBJECTIVE: New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. METHODS: Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. RESULTS: Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. CONCLUSION: This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies.

Ultrasound-guided noninvasive pancreas ablation using histotripsy: feasibility study in an in vivo porcine model.

Pancreatic cancer is a malignant disease associated with poor survival and nearly 80% present with unresectable tumors. Treatments such as chemotherapy and radiation therapy have shown overall improved survival benefits, albeit limited. Histotripsy is a noninvasive, non-ionizing, and non-thermal focused ultrasound ablation modality that has shown efficacy in treating hepatic tumors and other malignancies. In this novel study, we investigate histotripsy for noninvasive pancreas ablation in a pig model. In two studies, histotripsy was applied to the healthy pancreas in 11 pigs using a custom 32-element, 500 kHz histotripsy transducer attached to a clinical histotripsy system, with treatments guided by real-time ultrasound imaging. A pilot study was conducted in 3 fasted pigs with histotripsy applied at a pulse repetition frequency (PRF) of 500 Hz. Results showed no pancreas visualization on coaxial ultrasound imaging due to overlying intestinal gas, resulting in off-target injury and no pancreas damage. To minimize gas, a second group of pigs (n = 8) were fed a custard diet containing simethicone and bisacodyl. Pigs were euthanized immediately (n = 4) or survived for 1 week (n = 4) post-treatment. Damage to the pancreas and surrounding tissue was characterized using gross morphology, histological analysis, and CT imaging. Results showed histotripsy bubble clouds were generated inside pancreases that were visually maintained on coaxial ultrasound (n = 4), with 2 pigs exhibiting off-target damage. For chronic animals, results showed the treatments were well-tolerated with no complication signs or changes in blood markers. This study provides initial evidence suggesting histotripsy's potential for noninvasive pancreas ablation and warrants further evaluation in more comprehensive studies.

Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study.

Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.

NLRX1 functions as a tumor suppressor in Pan02 pancreatic cancer cells.

Pancreatic cancer is a deadly malignancy with limited treatment options. NLRX1 is a unique, understudied member of the Nod-like Receptor (NLR) family of pattern recognition receptors that regulates a variety of biological processes that are highly relevant to pancreatic cancer. The role of NLRX1 in cancer remains highly enigmatic, with some studies defining its roles as a tumor promoter, while others characterize its contributions to tumor suppression. These seemingly contradicting roles appear to be due, at least in part, to cell type and temporal mechanisms. Here, we define roles for NLRX1 in regulating critical hallmarks of pancreatic cancer using both gain-of-function and loss-of-function studies in murine Pan02 cells. Our data reveals that NLRX1 increases susceptibility to cell death, while also suppressing proliferation, migration, and reactive oxygen species production. We also show that NLRX1 protects against upregulated mitochondrial activity and limits energy production in the Pan02 cells. Transcriptomics analysis revealed that the protective phenotypes associated with NLRX1 are correlated with attenuation of NF-κB, MAPK, AKT, and inflammasome signaling. Together, these data demonstrate that NLRX1 diminishes cancer-associated biological functions in pancreatic cancer cells and establishes a role for this unique NLR in tumor suppression.

BTYNB, an inhibitor of RNA binding protein IGF2BP1 reduces proliferation and induces differentiation of leukemic cancer cells.

Leukemia is a group of diseases characterized by altered growth and differentiation of lymphoid or myeloid progenitors of blood. The existence of specific clusters of cells with stemness-like characteristics like differentiation, self-renewal, detoxification, and resistance to apoptosis in Leukemia makes them difficult to treat. It was recently reported that an oncofetal RNA binding protein, insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), maintains leukemic stem cell properties. BTYNB is an inhibitor of IGF2BP1 that was shown to affect the biological functions of IGF2BP1 however, the effect of BTYNB in Leukemia is not properly established. In this study, we assessed the effect of BTYNB on leukemic cell differentiation and proliferation. We performed cell viability assay to assess the effect of BTYNB in leukemic cells. We then assessed cell morphology of the leukemic cells treated with BTYNB. Further, we conducted an apoptosis assay and cell cycle assay. We found the cell viability of leukemic cells was significantly decreased post treatment with BTYNBs. Further, a noticeable morphological change was observed in BTYNB treated leukemic cells. BTYNB treated leukemic cells showed increased cell death and cell cycle arrest at S-phase. Evidence from the upregulation of BAK and p21 further confirmed apoptosis and cycle arrest. The gene expression of differentiation genes such as CD11B, ZFPM1, and KLF5 were significantly upregulated in BTYNB treated leukemic cells, therefore, confirming cell differentiation. Collectively, our study showed inhibition of IGF2BP1 function using BTYNB promotes differentiation in leukemic cells.

Exploration of metabolic responses towards hypoxia mimetic DMOG in cancer cells by using untargeted metabolomics.

Hypoxia is considered as one of the most crucial elements of tumor microenvironment. The hypoxia inducible transcription factors (HIF-1/2) are used by the cancer cells to adapt hypoxic microenvironment through regulating the expression of various target genes, including metabolic enzymes. Dimethyloxalylglycine (DMOG), a hypoxic mimetic used for HIF stabilisation in cell and animal models, also demonstrates multiple metabolic effects. In past, it was shown that in cancer cells, DMOG treatment alters mitochondrial ATP production, glycolysis, respiration etc. However, a global landscape of metabolic level alteration in cancer cells during DMOG treatment is still not established. In the current work, the metabolic landscape of cancer cells during DMOG treatment is explored by using untargeted metabolomics approach. Results showed that DMOG treatment primarily alters the one carbon and lipid metabolism. The levels of one-carbon metabolism related metabolites like serine, ornithine, and homomethionine levels significantly altered during DMOG treatment. Further, DMOG treatment reduces the global fatty acyls like palmitic acids, stearic acids, and arachidonic acid levels in cancer cell lines. Additionally, we found an alteration in glycolytic metabolites known to be regulated by hypoxia in cancer cell lines. Collectively, the results provided novel insights into the metabolic impact of DMOG on cancer cells and showed that the use of DMOG to induce hypoxia yields similar metabolic features relative to physiological hypoxia.

Epigenomic interplay in tumor heterogeneity: Potential of epidrugs as adjunct therapy.

"Heterogeneity" in tumor mass has immense importance in cancer progression and therapy. The impact of tumor heterogeneity is an emerging field and not yet fully explored. Tumor heterogeneity is mainly considered as intra-tumor heterogeneity and inter-tumor heterogeneity based on their origin. Intra-tumor heterogeneity refers to the discrepancy within the same cancer mass while inter-tumor heterogeneity refers to the discrepancy between different patients having the same tumor type. Both of these heterogeneity types lead to variation in the histopathological as well as clinical properties of the cancer mass which drives disease resistance towards therapeutic approaches. Cancer stem cells (CSCs) act as pinnacle progenitors for heterogeneity development along with various other genetic and epigenetic parameters that are regulating this process. In recent times epigenetic factors are one of the most studied parameters that drive oxidative stress pathways essential during cancer progression. These epigenetic changes are modulated by various epidrugs and have an impact on tumor heterogeneity. The present review summarizes various aspects of epigenetic regulation in the tumor microenvironment, oxidative stress, and progression towards tumor heterogeneity that creates complications during cancer treatment. This review also explores the possible role of epidrugs in regulating tumor heterogeneity and personalized therapy against drug resistance.

Exploration of Novel Pathways Underlying Irreversible Electroporation Induced Anti-Tumor Immunity in Pancreatic Cancer.

Advancements in medical sciences and technologies have significantly improved the survival of many cancers; however, pancreatic cancer remains a deadly diagnosis. This malignancy is often diagnosed late in the disease when metastases have already occurred. Additionally, the location of the pancreas near vital organs limits surgical candidacy, the tumor's immunosuppressive environment limits immunotherapy success, and it is highly resistant to radiation and chemotherapy. Hence, clinicians and patients alike need a treatment paradigm that reduces primary tumor burden, activates systemic anti-tumor immunity, and reverses the local immunosuppressive microenvironment to eventually clear distant metastases. Irreversible electroporation (IRE), a novel non-thermal tumor ablation technique, applies high-voltage ultra-short pulses to permeabilize targeted cell membranes and induce cell death. Progression with IRE technology and an array of research studies have shown that beyond tumor debulking, IRE can induce anti-tumor immune responses possibly through tumor neo-antigen release. However, the success of IRE treatment (i.e. full ablation and tumor recurrence) is variable. We believe that IRE treatment induces IFNγ expression, which then modulates immune checkpoint molecules and thus leads to tumor recurrence. This indicates a co-therapeutic use of IRE and immune checkpoint inhibitors as a promising treatment for pancreatic cancer patients. Here, we review the well-defined and speculated pathways involved in the immunostimulatory effects of IRE treatment for pancreatic cancer, as well as the regulatory pathways that may negate these anti-tumor responses. By defining these underlying mechanisms, future studies may identify improvements to systemic immune system engagement following local tumor ablation with IRE and beyond.

Experimental and Numerical Investigation of Parameters Affecting High-Frequency Irreversible Electroporation for Prostate Cancer Ablation.

While the primary goal of focal therapy for prostate cancer (PCa) is conserving patient quality of life by reducing oncological burden, available modalities use thermal energy or whole-gland radiation which can damage critical neurovascular structures within the prostate and increase risk of genitourinary dysfunction. High-frequency irreversible electroporation (H-FIRE) is a promising alternative ablation modality that utilizes bursts of pulsed electric fields (PEFs) to destroy aberrant cells via targeted membrane damage. Due to its nonthermal mechanism, H-FIRE offers several advantages over state-of-the-art treatments, but waveforms have not been optimized for treatment of PCa. In this study, we characterize lethal electric field thresholds (EFTs) for H-FIRE waveforms with three different pulse widths as well as three interpulse delays in vitro and compare them to conventional irreversible electroporation (IRE). Experiments were performed in non-neoplastic and malignant prostate cells to determine the effect of waveforms on both targeted (malignant) and adjacent (non-neoplastic) tissue. A numerical modeling approach was developed to estimate the clinical effects of each waveform including extent of nonthermal ablation, undesired thermal damage, and nerve excitation. Our findings indicate that H-FIRE waveforms with pulse durations of 5 and 10 µs provide large ablations comparable to IRE with tolerable levels of thermal damage and minimized muscle contractions. Lower duration (2 µs) H-FIRE waveforms exhibit the least amount of muscle contractions but require increased voltages which may be accompanied by unwanted thermal damage.

Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review.

New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.

Properties of tissue within prostate tumors and treatment planning implications for ablation therapies.

Irreversible electroporation (IRE) is a promising alternative therapy for the local treatment of prostate tumors. The procedure involves the direct insertion of needle electrodes into the target zone, and subsequent delivery of short but high-voltage pulses. Successful outcomes rely on adequate exposure of the tumor to a threshold electrical field. To aid in predicting this exposure, computational models have been developed, yet often do not incorporate the appropriate tissue-specific properties. This work aims to quantify electrical conductivity behavior during IRE for three types of tissue present in the target area of a prostate cancer ablation: the tumor tissue itself, the surrounding healthy tissue, and potential areas of necrosis within the tumor. Animal tissues were used as a stand-in for primary samples. The patient-derived prostate tumor tissue showed very similar responses to healthy porcine prostate tissue. An examination of necrotic tissue inside the tumors revealed a large difference, however, and a computational model showed that a necrotic core with differing electrical properties can cause unexpected inhomogeneities within the treatment region.

Generation of Tumor-activated T cells Using Electroporation.

Expansion of cytotoxic T lymphocytes (CTLs) is a crucial step in almost all cancer immunotherapeutic methods. Current techniques for expansion of tumor-reactive CTLs present major limitations. This study introduces a novel method to effectively produce and expand tumor-activated CTLs using high-voltage pulsed electric fields. We hypothesize that utilizing high-voltage pulsed electric fields may be an ideal method to activate and expand CTLs due to their non-thermal celldeath mechanism. Tumor cells were subjected to high-frequency irreversible electroporation (HFIRE) with various electric field magnitudes (1250, 2500 V/cm) and pulse widths (1, 5, and 10 µs), or irreversible electroporation (IRE) at 1250 V/cm. The treated tumor cells were subsequently cocultured with CD4+ and CD8+ T cells along with antigen-presenting cells. We show that tumor-activated CTLs can be produced and expanded when exposed to treated tumor cells. Our results suggest that CTLs are more effectively expanded when pulsed with HFIRE conditions that induce significant cell death (longer pulse widths and higher voltages). Activated CD8+ T cells demonstrate cytotoxicity to untreated tumor cells suggesting effector function of the activated CTLs. The activated CTLs produced with our technique could be used for clinical applications with the goal of targeting and eliminating the tumor.

Biochanin A induces a brown-fat phenotype via improvement of mitochondrial biogenesis and activation of AMPK signaling in murine C3H10T1/2 mesenchymal stem cells.

In this study, we investigated the effect of Biochanin A (BioA), an O-methylated isoflavone on the brown-fat phenotype formation and on the associated thermogenic program including mitochondrial biogenesis and lipolysis in C3H10T1/2 MSCs. Our data demonstrates that Treatment with BioA in an adipogenic differentiation cocktail induced formation of brown-fat-like adipocytes from C3H10T1/2 MSCs without treatment with a known browning inducer (rosiglitazone or T3) at an early stage of differentiation. The formation of brown-fat-like adipocytes by BioA treatment was evidenced by upregulation of key thermogenic markers: Ucp1, Pgc1α, Prdm16, and Pparγ. BioA also increased the expression of beige (Cd137 and Fgf21) and brown (Elovl3 and Zic1)-specific markers. Additionally, BioA treatment promoted mitochondrial biogenesis, judging by the upregulation of genes; Cox8b, Cidea, Dio2, Sirt1, Opa1, and Fis1. BioA treatment increased the amount of mitochondrial DNA and its encoded proteins: oxidative phosphorylation complexes (I-V); this change was associated with high oxygen consumption by C3H10T1/2 MSCs. A small-interfering-RNA-induced gene knockdown and experiments with dorsomorphin-driven competitive inhibition revealed that BioA exerts the thermogenic action via activation of AMPK signaling. Our study shows the mechanism of BioA-induced promotion of a brown-fat phenotype. Nonetheless, clinical research is necessary to validate BioA as a brown-fat-like signature inducer.

Sinapic acid induces the expression of thermogenic signature genes and lipolysis through activation of PKA/CREB signaling in brown adipocytes.

Lipid accumulation in white adipose tissue is the key contributor to the obesity and orchestrates numerous metabolic health problems such as type 2 diabetes, hypertension, atherosclerosis, and cancer. Nonetheless, the prevention and treatment of obesity are still inadequate. Recently, scientists found that brown adipose tissue (BAT) in adult humans has functions that are diametrically opposite to those of white adipose tissue and that BAT holds promise for a new strategy to counteract obesity. In this study, we evaluated the potential of sinapic acid (SA) to promote the thermogenic program and lipolysis in BAT. SA treatment of brown adipocytes induced the expression of brown-adipocyte activation-related genes such as Ucp1, Pgc-1α, and Prdm16. Furthermore, structural analysis and western blot revealed that SA upregulates protein kinase A (PKA) phosphorylation with competitive inhibition by a pan-PKA inhibitor, H89. SA binds to the adenosine triphosphate (ATP) site on the PKA catalytic subunit where H89 binds specifically. PKA-cat-α1 gene-silencing experiments confirmed that SA activates the thermogenic program via a mechanism involving PKA and cyclic AMP response element-binding protein (CREB) signaling. Moreover, SA treatment promoted lipolysis via a PKA/p38-mediated pathway. Our findings may allow us to open a new avenue of strategies against obesity and need further investigation. [BMB Reports 2020; 53(3): 142-147].

Medicarpin induces lipolysis via activation of Protein Kinase A in brown adipocytes.

Natural pterocarpan Medicarpin (Med) has been shown to have various beneficial biological roles, including inhibition of osteoclastogenesis, stimulation of bone regeneration and induction of apoptosis. However, the effect of the Med on lipolysis in adipocytes has not been reported. Here, we show the effect of Med on lipolysis in different mouse adipocytes and elucidate the underlying mechanism. We observed that Med treatment promoted release of glycerol in the media. Differentiated mouse brown adipose tissue cells were treated with Med. RNA-Seq analysis was performed to elucidate the effect of med and subsequently was confirmed by qRT-PCR and western blotting analyses. Med treatment increased both protein and gene expression levels of hormone-sensitive lipase (Hsl) and adipose triglyceride lipase (Atgl), which are two critical enzymes necessary for lipolysis. Mechanistic study showed that Med activates Protein Kinase A (PKA) and phosphorylates Hsl at PKA target position at Serine660. Silencing of PKA gene by short interfering RNA attenuated the Med-induced increase in glycerol release and Hsl phosphorylation. The results unveil that Med boosts lipolysis via a PKA-dependent pathway in adipocytes and may provide a possible avenue of further research of Med mediated reduction of body fat. [BMB Reports 2018; 51(5): 249-254].