Disruption Of Calcium Homeostasis Research Articles

Inhibition of TBL1 cleavage alleviates doxorubicin-induced cardiomyocytes death by regulating the Wnt/β-catenin signal pathway

Abstract Aims Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. Methods and results We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy (DCM) and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites—D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with β-catenin. As a result, Wnt reporter activity, and Wnt target gene expression collectively indicate a decrease in Wnt/β-catenin signaling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. Conclusions Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Endoplasmic reticulum targeted fluorescent probe for real-time monitoring the viscosity changes induced by calcium homeostasis disruption

The endoplasmic reticulum (ER) acts as the major storage site for calcium ions, which are messenger ions for intracellular signaling. Disruption of calcium ion homeostasis can significantly affect the viscosity, polarity and pH of the ER. However, it is still unclear the relationship between the viscosity changes in ER and the imbalance of calcium ion homeostasis. Herein, we developed a novel fluorescent probe, named TPA, for monitoring viscosity changes that specifically targets the endoplasmic reticulum rather than mitochondria or lysosomes. TPA probe displayed good stability, as well as high responsiveness and selectivity to viscosity. The fluorescence intensity of TPA was significantly enhanced with the increased concentration or incubation time of the stimulating agents(i.e., tunicamycin), showing high responsiveness to the viscosity changes in ER. Furthermore, the TPA probe successfully demonstrated that an increase in intracellular calcium ion concentration leads to an increase in ER viscosity, whereas a decrease in calcium ion concentration leads to a decrease viscosity in ER. Both in vitro and in vivo experiments demonstrated that TPA probe successfully detected the viscosity changes in ER, especially the effects of calcium ion homeostasis disruption on ER. Overall, the TPA probe represents an efficient method for studying the relationship between calcium ion homeostasis and ER viscosity.

Protective effects of arbutin against doxorubicin-induced cardiac damage.

Doxorubicin is an effective antineoplastic agent but has limited clinical application because of its cumulative toxicities, including cardiotoxicity. Cardiotoxicity causes lipid peroxidation, genetic impairment, oxidative stress, inhibition of autophagy, and disruption of calcium homeostasis. Doxorubicin-induced cardiotoxicity is frequently tried to be mitigated by phytochemicals, which are derived from plants and possess antioxidant, anti-inflammatory, and anti-apoptotic properties. Arbutin, a natural antioxidant found in the leaves of the bearberry plant, has numerous pharmacological benefits, including antioxidant, anti-bacterial, anti-hyperglycemic, anti-inflammatory, and anti-tumor activity. The study involved male Wistar rats divided into three groups: a control group, a group treated with doxorubicin (20mg/kg) to induce cardiac toxicity, a group treated with arbutin (100mg/kg) daily for two weeks before doxorubicin administration. After treatment, plasma and heart tissue samples were collected for analysis. The samples were evaluated for oxidative stress parameters, including superoxide dismutase, malondialdehyde, and catalase, as well as for cardiac biomarkers, including CK, CK-MB, and LDH. The heart tissues were also analyzed using molecular (TNF-α, IL-1β and Caspase 3), histopathological and immunohistochemical methods (8-OHDG, 4 Hydroxynonenal, and dityrosine). The results showed that arbutin treatment was protective against doxorubicin-induced oxidative damage by increasing SOD and CAT activity and decreasing MDA level. Arbutin treatment was similarly able to reverse the inflammatory response caused by doxorubicin by reducing TNF-α and IL-1β levels and also reverse the apoptosis by decreasing caspase-3 levels. It was able to prevent doxorubicin-induced cardiac damage by reducing cardiac biomarkers CK, CK-MB and LDH levels. In addition to all these results, histopathological analyzes also show that arbutin may be beneficial against the damage caused by doxorubicin on heart tissue. The study suggests that arbutin has the potential to be used to mitigate doxorubicin-induced cardiotoxicity in cancer patients.

Paucatalinone A from Paulownia Catalpifolia Gong Tong Elicits mitochondrial-mediated cancer cell death to combat osteosarcoma.

As the global cancer burden escalates, the search for alternative therapies becomes increasingly vital. Natural products, particularly plant-derived compounds, have emerged as promising alternatives to conventional cancer treatments due to their diverse bioactivities and favorable biosafety profiles. Here, we investigate Paucatalinone A, a newly discovered geranylated flavanone derived from the fruit of Paulownia Catalpifolia Gong Tong, notable for its significant anti-cancer properties. We revealed the capability of Paucatalinone A to induce apoptosis in osteosarcoma cells and deciphered its underlying mechanisms. Our findings demonstrate that Paucatalinone A substantially augments apoptosis, inhibits cell proliferation, and demonstrates a pronounced anti-tumor effect in a murine model of osteosarcoma. Mechanistically, Paucatalinone A disrupts calcium homeostasis and exacerbates intracellular reactive oxygen species accumulation, leading to mitochondrial impairment, cytoskeletal collapse, and caspase-dependent apoptotic cell death. This study underscores the potential of Paucatalinone A in initiating apoptosis in cancer cells and highlights the therapeutic efficacy of plant-derived agents in treating osteosarcoma, offering a viable approach for managing other intractable cancers.

Mechanistic insights into the antitumoral potential and in vivo antiproliferative efficacy of a silver-based core@shell nanosystem

This study delves into the biomolecular mechanisms underlying the antitumoral efficacy of a hybrid nanosystem, comprised of a silver core@shell (Ag@MSNs) functionalized with transferrin (Tf). Employing a SILAC proteomics strategy, we identified over 150 de-regulated proteins following exposure to the nanosystem. These proteins play pivotal roles in diverse cellular processes, including mitochondrial fission, calcium homeostasis, endoplasmic reticulum (ER) stress, oxidative stress response, migration, invasion, protein synthesis, RNA maturation, chemoresistance, and cellular proliferation. Rigorous validation of key findings substantiates that the nanosystem elicits its antitumoral effects by activating mitochondrial fission, leading to disruptions in calcium homeostasis, as corroborated by RT-qPCR and flow cytometry analyses. Additionally, induction of ER stress was validated through western blotting of ER stress markers. The cytotoxic action of the nanosystem was further affirmed through the generation of cytosolic and mitochondrial reactive oxygen species (ROS). Finally, in vivo experiments using a chicken embryo model not only confirmed the antitumoral capacity of the nanosystem, but also demonstrated its efficacy in reducing cellular proliferation. These comprehensive findings endorse the potential of the designed Ag@MSNs-Tf nanosystem as a groundbreaking chemotherapeutic agent, shedding light on its multifaceted mechanisms and in vivo applicability.

Meptyldinocap induces implantation failure by forcing cell cycle arrest, mitochondrial dysfunction, and endoplasmic reticulum stress in porcine trophectoderm and endometrial luminal epithelial cells

Meptyldinocap is a dinitrophenol fungicide used to control powdery mildew. Although other dinitrophenol pesticides have been found to exhibit reproductive toxicity, studies of meptyldinocaps are scarce. This study investigated the adverse effects of meptyldinocap on porcine trophectoderm (pTr) and porcine endometrial luminal epithelial (pLE) cells, which play crucial roles in implantation. We confirmed that meptyldinocap decreased cell viability, induced apoptosis, and inhibited proliferation by decreasing proliferation-related gene expression and inducing changes in the cell cycle. Furthermore, meptyldinocap treatment caused mitochondrial dysfunction, endoplasmic reticulum stress, and disruption of calcium homeostasis. Moreover, it induces alterations in mitogen-activated protein kinase signaling cascades and reduces the migration ability, leading to implantation failure. Our findings suggest that meptyldinocap reduces the cellular functions of pTr and pLE cells, which are important for the implantation process, and interferes with interactions between the two cell lines, potentially leading to implantation failure. We also propose a mechanism by which the understudied fungicide meptyldinocap exerts its cytotoxicity.

Serum albumin adjusted calcium and fluoride among type 2 diabetes mellitus patients.

The regulation of glucose-dependent insulin secretion in pancreatic β-cells relies on calcium concentration, making it a calcium-dependent process. Disruptions in calcium homeostasis may contribute to insulin dysfunction and disturbances in glucose homeostasis, potentially playing a role in the development of type 2 diabetes mellitus (T2DM). In T2DM patients, there may be changes in fluoride metabolism due to impaired renal function. Measurement of serum albumin-adjusted calcium and is crucial since changes in albumin levels can impact the accurate estimation of true calcium.

Nanoengineering Calcium Peroxide‐Based Site‐Specific Delivery Platform to Efficiently Activate the cGAS‐STING Pathway for Cancer Immunotherapy by Amplified Endoplasmic Reticulum Stress

Abstract Currently, the understanding of the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) pathway's involvement in efficient immunotherapy mainly revolves around the role of mitochondria or nucleus modulation. Nonetheless, the role of endoplasmic reticulum (ER) stress in activating the cGAS‐STING mechanism to boost immunity against tumors remains essentially unexplored. Herein, novel findings demonstrating that ER stress can be used as a strategy for stimulating the cGAS‐STING pathway, thereby augmenting the immune response against cancer, are presented. To accomplish this objective, ER‐targeting p‐methylbenzene sulfonamide‐tailored IR780 (p‐780) is synthesized and it is loaded into CaO2 nanoparticles, which are further functionalized with distearoyl phosphoethanolamine‐polyethylene glycol（DSPE‐PEG）‐biotin to form PEG/CaO2@p‐780 NPs. The disruption of calcium homeostasis, coupled with the heightened levels of reactive oxygen species (ROS) mediated by p‐780, along with hyperpyrexia, collectively contributes to the amplification of endoplasmic reticulum (ER) stress. This cascade of events effectively triggers the cGAS‐STING pathway and, in parallel, facilitates the degradation of the programmed cell death 1 ligand 1 (PD‐L1) protein. In addition, oxygen released through CaO2 decomposition is expected to promote p‐780–mediated phototherapy, while reversing the immunosuppressive tumor microenvironment associated with hypoxia. Furthermore, DSPE‐PEG‐biotin facilitates tumor site‐specific drug delivery through active targeting mediated by the biotin receptor. Collectively, PEG/CaO2@p‐780 nanoparticles successfully activate systemic antitumor immunity by enhancing ER stress.

Metabolism-Regulating Nanozyme System for Advanced Nanocatalytic Cancer Therapy.

Nanocatalytic therapy, an emerging approach in cancer treatment, utilizes nanomaterials to initiate enzyme-mimetic catalytic reactions within tumors, inducing tumor-suppressive effects. However, the targeted and selective catalysis within tumor cells is challenging yet critical for minimizing the adverse effects. The distinctive reliance of tumor cells on glycolysis generates abundant lactate, influencing the tumor's pH, which can be manipulated to selectively activate nanozymatic catalysis. Herein, small interfering ribonucleic acid (siRNA) targeting lactate transporter-mediated efflux is encapsulated within the iron-based metal-organic framework (FeMOF) and specifically delivered to tumor cells through cell membrane coating. This approach traps lactate within the cell, swiftly acidifying the tumor cytoplasm and creating an environment for boosting the catalysis of the FeMOF nanozyme. The nanozyme generates hydroxyl radical (·OH) in the reversed acidic environment, using endogenous hydrogen peroxide (H2 O2 ) produced by mitochondria as a substrate. The induced cytoplasmic acidification disrupts calcium homeostasis, leading to mitochondrial calcium overload, resulting in mitochondrial dysfunction and subsequent tumor cell death. Additionally, the tumor microenvironment is also remodeled, inhibiting migration and invasion, thus preventing metastasis. This groundbreaking strategy combines metabolic regulation with nanozyme catalysis in a toxic drug-free approach for tumor treatment, holding promise for future clinical applications.

The role of calcium dynamics with amyloid beta on neuron-astrocyte coupling

Amyloid beta ($A\beta$) plaques are associated with neurodegenerative diseases such as Alzheimer's disease. Due to the involvement of $A\beta$ plaques in the functioning of the brain; cognitive decline disrupts calcium homeostasis in nerve cells and causes abnormal calcium ions ($Ca^{2+}$) signaling patterns. In consequence, there is enhanced neuronal excitability, compromised synaptic transmission, and decreased astrocytic function. Neuron-astrocyte coupling through calcium dynamics with different neuronal functions has been studied. Key signaling molecules in this process include $Ca^{2+}$, which control several cellular functions, including neurotransmission and astrocytic regulation. The mathematical model for neuron-astrocyte communication has been developed to study the importance of calcium dynamics in signal transduction between the cells. To understand the wide role of mitochondria, NCX, and amyloid beta with various necessary parameters included in the model, $Ca^{2+}$ signaling patterns have been analyzed through amplitude modulation and frequency modulation. The results of the current model are simulated and analyzed using XPPAUT. The findings of the current study are contrasted with experimental data from an existing mathematical model that illustrates the impact of calcium oscillation frequency and amplitude modulations in nerve cells.

Cognitive decline and affective alterations in SCA2-58Q mice

The cerebellum is traditionally recognized as a brain region dedicated to motor control and movement coordination. Yet, the growing body of research has recently highlighted the cerebellar role in cognitive processes, learning, and emotional states. Patients with damage to this particular region of the brain exhibit deficiencies in verbal communication, impaired speech, executive functions, and memory, along with various affective disorders [1]. In the field of neurodegeneration research, the cerebellum is primarily recognized for its role in the onset of spinocerebellar ataxias (SCAs). SCA2 is an autosomal dominant polyglutamine-related neurological disorder resulting from a notable escalation in the number of CAG triplet repeats that encode the amino acid glutamine in the ATXN2 gene. The mutated protein ataxin-2 fails to perform its molecular functions, disrupting calcium homeostasis and cellular activity, ultimately resulting in widespread Purkinje neuron death in the cerebellar cortex [2]. In our research, we used SCA2-58Q transgenic mice that express the mutant human ataxin-2 gene under the action of the Purkinje-specific promoter (PCP2/L7). Each of these tests were aimed at evaluating specific aspects of the mice’s behavior. This model enabled to investigate the direct involvement of Purkinje cells in the cognitive and affective symptoms associated with SCA2. Our team conducted three behavioral tests to determine the anxiolytic behavior: the open field test, the novelty suppressed feeding test, and the light-dark place preference test. The novel object recognition and fear conditioning tests were used to assess recognition and contextual memory, respectively. The Morris water maze test was conducted to evaluate spatial learning and memory. The researchers assessed depression and anhedonia levels using the forced swimming, tail suspension, and sucrose preference tests. The study revealed anxiety, spatial memory impairment, and affective decline in 7–8-month-old SCA2 mice. Previous results of the beam walk test [3, 4] indicated a delay in motor skill development in SCA2-58Q mice, which began at 8–10 months of age. This indicates that cognitive decline and affective changes occur before the primary motor impairment in spinocerebellar ataxia type 2. Currently, there is no disease-modifying treatment for SCA2, and patients are typically given supportive therapy and treatment for individual symptoms. In conclusion, our research provides a more comprehensive clinical picture and takes a step towards optimizing the diagnosis and selecting more effective therapy options for SCA2.

Calcium Activation of the Androgen Receptor in Prostate Cells.

Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor. The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured. To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels. The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

Alcohol induces ER stress and apoptosis by inducing oxidative stress and disruption of calcium homeostasis in glial cells

Alcohol has teratogenic effects that can cause developmental abnormalities and alter anatomical and functional characteristics of the developed brain and other organs. Glial cells play a crucial role in alcohol metabolism and protect neurons from toxic effects of alcohol. However, chronic alcohol exposure can lead to uncontrollable levels of reactive oxygen species, resulting in the death of glial cells and exposing neuronal cells to the toxic effects of alcohol. The exact molecular mechanism of alcohol-induced glial cell death has not been fully explored. This study reported that different concentrations of alcohol induce different expressions of ER stress markers in glial cells, focusing on the role of endoplasmic reticulum (ER) stress. Alcohol-induced concentration-dependent toxicity in both cells also induced oxidative stress, leading to mitochondrial damage. The expression of p53 and apoptotic proteins was significantly up-regulated after alcohol exposure, while Bcl2 (anti-apoptotic) was down-regulated. The signalling pathway for ER stress was activated and up-regulated marker proteins in a concentration-dependent manner. Cells pre-treated with BAPTA-AM and NAC showed significant resistance against alcohol assault compared to other cells. These in vitro findings will prove valuable for defining the mechanism by which alcohol modulates oxidative stress, mitochondrial and ER damage leading to glial cell death.

Elevation of CISD2 attenuates atrial aging

Abstract Background Age-related atrial cardiomyopathy is characterized by conduction disturbance, structural fibrosis as well as cardiac remodeling. Atrial cardiomyopathy promotes the development of atrial fibrillation, which is associated with a 5-fold increase in the risk of stroke. However, the molecular mechanisms underlying age-associated deterioration of atrial function remain unclear. Cisd2 is a mitochondrial outer membrane protein that regulates intracellular calcium homeostasis and mitochondrial integrity. Importantly, Cisd2 is a pro-longevity gene in mammals. Purpose In this study, we investigate the role of Cisd2 in atrial cardiomyopathy during aging. Methods CISD2 expression and atrial conductance were examined in different age human surgical samples. Atrial conductance was assessed and validated by immunofluorescence and Transmission electron microscope in Cisd2 knockout and overexpressing transgenic mice at young and old age. The mitochondrial functions and calcium homeostasis were investigated in the atrial HL-1 cell lines with Cisd2KO or re-expression. Transcriptomic analysis of mice atrial tissues was performed. Results The age-associated decrease of atrial Cisd2 protein, in human and mice, were associated with atrial conductance disturbance and intercalated disc disorganization. Furthermore, Cisd2 deficiency disrupts calcium homeostasis via dysregulation of Serca2a and Stim1 resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in HL-1 atrial cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay age-associated atrial myopathy and attenuate age-related atrial structural defects and functional decline. In addition, it results in a younger atrial transcriptome pattern during old age. Conclusions The mitochondrial-associated membrane-bounded CDGSH iron-sulfur domain-containing protein 2 (CISD2) protein regulates cytosolic calcium homeostasis and mitochondrial integrity. Interestingly, an age-dependent decrease of atrial Cisd2 protein has been detected during aging in both humans and mice. Our findings indicate that decreased atrial Cisd2 is one of the causes of atrial myopathy during cardiac aging. A persistently high level of atrial Cisd2 delays atrial myopathy during aging and ameliorates age-related atrial dysfunction.

THU396 Digital Spatial Profiling Of Human Parathyroid Tumors Reveals Distinct Cellular And Molecular Alterations Linked To Vitamin D Deficiency

Abstract Disclosure: C. Tu: None. W. Chang: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly & Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly & Company. J. Koh: None. Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population, but the pathophysiological basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from 9 vitamin D-deficient or 34 vitamin D-replete PHPT patients with 12 eucalcemic cadaveric donor parathyroid glands as normal tissue controls. Parathyroid oxyphil cell content is markedly higher in tumors from vitamin D-deficient PHPT patients (Def-Ts) relative to tumors from vitamin D-replete PHPT patients (Rep-Ts) (47.8% vs 17.8%, respectively; p <0.0001) and normal donor parathyroid glands (7.7%). Def-Ts display increased expression of genes encoding electron transport chain (corr. coefficient=0.546), proteasomal catabolism (corr. coefficient=0.587), and oxidative phosphorylation (corr. coefficient=0.982) pathway components. In contrast, Rep-Ts are characterized by upregulation of the ras (corr. coefficient=0.632) and myc (corr. coefficient=0.811) signaling pathways, suggestive of an oncogene-driven etiology. Parathyroid oxyphil cells are comparable to chief cells at the transcriptional level, and vitamin D-status is reflected in the transcriptional profiles of both cell types in a similar manner. Among PHPT patients who did not present as vitamin D deficient, three distinct tumor transcriptional profile clusters showed a dose-dependent relationship to pre-operative 25OHD levels (p <0.0001 by ANOVA). Relative to normal tissue, the abundance of calcium sensing receptor (CaSR) protein is reduced in Def-Ts (mean protein count 253.6 vs 492.3, respectively; p=0.039 for difference), while the type B γ-aminobutyric acid receptor 1 (GABAB1R) is increased (mean protein count 114.5 vs 64.65, respectively; p=0.0099 for difference). These expression changes resulted in a vitamin D dose-dependent reduction in the ratio of CaSR to GABAB1R receptors in Def-T tumor cells (r2 = 0.218, p<0.0001), favoring the formation of calcium sensing-attenuated CaSR:GABAB1R heteromers. The abundance of the amyloid precursor protein (APP) peptide derivative Aβ1-42, a putative GABAB1R ligand, is preferentially increased in Def-Ts compared to normal tissue (mean protein count 5797 vs 4500, respectively; p=0.0318). Collectively, these findings suggest a novel mechanism where chronic vitamin D deficiency can contribute to tonic PTH hypersecretion in PHPT patients by increasing GABAB1R-mediated antagonism of CaSR activity in parathyroid adenomas. Presentation: Thursday, June 15, 2023

KCTD4 interacts with CLIC1 to disrupt calcium homeostasis and promote metastasis in esophageal cancer.

Increasing evidences suggest the important role of calcium homeostasis in hallmarks of cancer, but its function and regulatory network in metastasis remain unclear. A comprehensive investigation of key regulators in cancer metastasis is urgently needed. Transcriptome sequencing (RNA-seq) of primary esophageal squamous cell carcinoma (ESCC) and matched metastatic tissues and a series of gain/loss-of-function experiments identified potassium channel tetramerization domain containing 4 (KCTD4) as a driver of cancer metastasis. KCTD4 expression was found upregulated in metastatic ESCC. High KCTD4 expression is associated with poor prognosis in patients with ESCC and contributes to cancer metastasis invitro and invivo. Mechanistically, KCTD4 binds to CLIC1 and disrupts its dimerization, thus increasing intracellular Ca2+ level to enhance NFATc1-dependent fibronectin transcription. KCTD4-induced fibronectin secretion activates fibroblasts in a paracrine manner, which in turn promotes cancer cell invasion via MMP24 signaling as positive feedback. Furthermore, a lead compound K279-0738 significantly suppresses cancer metastasis by targeting the KCTD4‒CLIC1 interaction, providing a potential therapeutic strategy. Taken together, our study not only uncovers KCTD4 as a regulator of calcium homeostasis, but also reveals KCTD4/CLIC1-Ca2+-NFATc1-fibronectin signaling as a novel mechanism of cancer metastasis. These findings validate KCTD4 as a potential prognostic biomarker and therapeutic target for ESCC.

Primary choledocholithiasis occurrence and recurrence is synergetcally modulated by the bile microbiome and metabolome alternations

AimsPrimary choledocholithiasis is a common digestive disease with high morbidity and relapse. However, the compositions and functions of the bile microbial ecosystem and the pathogenesis of microfloral regulation of host metabolism resulting in stone formation are poorly understood. Main methodsBiliary samples collected from patients with acute cholangitis induced by benign biliary stricture (nonlithiasis group, n = 17) and primary choledocholithiasis (lithiasis group, n = 33) were subjected to multiomics analyses. Furthermore, clinicopathological features collected over a 24-month follow-up period were examined to evaluate the predictive value of candidate microbes. Key findingsFive alpha diversity indices of the bile microbiome were significantly decreased in the lithiasis group. Furthermore, we identified 49 differential bile flora between the two groups, and the relative abundances of 6 bacteria, Actinobacteria, Actinobacteriota, Staphylococcales, Micrococcales, Altererythrobacter and Carnobacteriaceae, were associated with primary choledocholithiasis relapse conditions. Multiomics analyses showed that specific changes in disease-related bacterial taxa were closely related to metabolite variation (low-molecular weight carboxylic acids, sterol liquid and acylcarnitine), which might reflect disease prognosis. According to microbiomic and metabolomic pathway analyses, we revealed that bacterial infections, microbiota-derived amino acid metabolites and secondary bile acid-related pathways were significantly enriched in the stone-formation group, suggesting a novel host-microbial metabolic mechanism of primary choledocholithiasis. SignificanceOur study first indicates bile host-microbial dysbiosis modulates the abnormal accumulation of metabolites might further disrupt calcium homeostasis and generate insoluble saponification. Additionally, we determined the predictive value of Actinomycetes phylum reduction for recurrence in primary common bile duct stone patients.

Decoding the biological toxicity of phenanthrene on intestinal cells of Eisenia fetida: Effects, toxicity pathways and corresponding mechanisms

Phenanthrene is frequently detected and exists extensively in the soil environment, and its residues inevitably impose a significant threat to soil organisms. Exposure to and toxicity of phenanthrene on earthworms has been extensively studied before, however, the possible mechanisms and related pathways associated with phenanthrene-triggered toxicity at the intestinal cell level remain unclear. Herein, primary intestinal cells isolated from Eisenia fetida (Annelida, Oligochaeta) intestine were used as targeted receptors to probe the molecular mechanisms involved in ROS-mediated damaging effects and the potential pathways of phenanthrene-induced toxicity at cellular and sub-cellular levels. Results indicated that phenanthrene exposure induced oxidative stress by activating intracellular ROS (elevated O2−, H2O2, and OH− content) bursts in E. fetida intestinal cells, causing various oxidative damage effects, including lipid peroxidation (increased MDA content), protein oxidation (enhanced PCO levels), and DNA damage (enhanced 8-OHdG levels). The enzymatic and non-enzymatic strategies in earthworm cells were activated to mitigate these detrimental effects by regulating ROS-mediated pathways involving defense regulation. Also, phenanthrene stress destroyed the cell membrane of E. fetida intestinal cells, resulting in cellular calcium homeostasis disruption and cellular energetic alteration, ultimately causing cytotoxicity and cell apoptosis/death. More importantly, the mitochondrial dysfunction in E. fetida cells was induced by phenanthrene-caused mitochondrial membrane depolarization, which in turn caused un-controlled ROS burst and induced apoptosis through mitochondria-mediated caspase-3 activation and ROS-mediated mitochondrial-dependent pathway. Furthermore, exposure to phenanthrene activated an abnormal mRNA expression profile associated with defense regulation (e.g., Hsp70, MT, CRT, SOD, CAT, and GST genes) in E. fetida intestinal cells, resulting in various cellular dysfunctions and pathological conditions, eventually, apoptotic cell death. Taken together, this study offers valuable insights for probing the toxic effects and underlying mechanisms posed by phenanthrene at the intestinal cell level, and is of great significance to estimate the detrimental side effects of phenanthrene on soil ecological health.

Effects of low doses of methylmercury (MeHg) exposure on definitive endoderm cell differentiation in human embryonic stem cells.

Fetal development is one of the most sensitive windows to methylmercury (MeHg) toxicity. Laboratory and epidemiological studies have shown a dose-response relationship between fetal MeHg exposure and neuro performance in different life stages from infants to adults. In addition, MeHg exposure has been reported to be associated with disorders in endoderm-derived organs, such as morphological changes in liver cells and pancreatic cell dysfunctions. However, the mechanisms of the effects of MeHg on non-neuronal organs or systems, especially during the early development of endoderm-derived organs, remain unclear. Here we determined the effects of low concentrations of MeHg exposure during the differentiation of definitive endoderm (DE) cells from human embryonic stem cells (hESCs). hESCs were exposed to MeHg (0, 10, 100, and 200nM) that covers the range of Hg concentrations typically found in human maternal blood during DE cell induction. Transcriptomic analysis showed that sub-lethal doses of MeHg exposure could alter global gene expression patterns during hESC to DE cell differentiation, leading to increased expression of endodermal genes/proteins and the over-promotion of endodermal fate, mainly through disrupting calcium homeostasis and generating ROS. Bioinformatic analysis results suggested that MeHg exerts its developmental toxicity mainly by disrupting ribosome biogenesis during early cell lineage differentiation. This disruption could lead to aberrant growth or dysfunctions of the developing endoderm-derived organs, and it may be the underlying mechanism for the observed congenital diseases later in life. Based on the results, we proposed an adverse outcome pathway for the effects of MeHg exposure during human embryonic stem cells to definitive endoderm differentiation.