Anlotinib Reverses Multidrug Resistance (MDR) in Osteosarcoma by Inhibiting P-Glycoprotein (PGP1) Function In Vitro and In Vivo

Overexpression of the multidrug resistance (MDR)-related protein P-glycoprotein (PGP1), which actively extrudes chemotherapeutic agents from cells and significantly decreases the efficacy of chemotherapy, is viewed as a major obstacle in osteosarcoma chemotherapy. Anlotinib, a novel tyrosine kinase inhibitor (TKI), has good anti-tumor effects in a variety of solid tumors. However, there are few studies on the mechanism of anlotinib reversing chemotherapy resistance in osteosarcoma. In this study, cellular assays were performed in vitro and in vivo to evaluate the MDR reversal effects of anlotinib on multidrug-resistant osteosarcoma cell lines. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The vanadate-sensitive ATPase activity of PGP1 was measured in the presence of a range of anlotinib concentrations. The protein expression level of ABCB1 was detected by Western blotting and immunofluorescence analysis. Our results showed that anlotinib significantly increased the sensitivity of KHOSR2 and U2OSR2 cells (which overexpress PGP1) to chemotherapeutic agents in vitro and in a KHOSR2 xenograft nude mouse model in vivo. Mechanistically, anlotinib increases the intracellular accumulation of PGP1 substrates by inhibiting the efflux function of PGP1 in multidrug-resistant cell lines. Furthermore, anlotinib stimulated the ATPase activity of PGP1 but affected neither the protein expression level nor the localization of PGP1. In animal studies, anlotinib in combination with doxorubicin (DOX) significantly decreased the tumor growth rate and the tumor size in the KHOSR2 xenograft nude mouse model. Overall, our findings suggest that anlotinib may be useful for circumventing MDR to other conventional antineoplastic drugs.

Conjugated Polyamines in Root Plasma Membrane Enhanced the Tolerance of Plum Seedling to Osmotic Stress by Stabilizing Membrane Structure and Therefore Elevating H+-ATPase Activity

Polyamines are small positively charged molecules in plants and play important functions in many biological processes under various environmental stresses. One of the most confounding problems relating to polyamines (PAs) in stresses is the lack of understanding of the mechanisms underlying their function(s). Furthermore, a limited number of studies have addressed this issue at the sub-cellular level, especially in tree plants under drought stress. Therefore, in this research, by simulating natural drought stress with polyethylene glycol (PEG) osmotic stress, the relationship between the levels of conjugated polyamines and the activity of H+-ATPase in the plasma membrane was elucidated with the roots of two plum (Prunus salicina L.) cultivars, which were different in drought tolerance, as experimental materials. Furthermore, free PA levels and the activities of S-adenosylmethionine decarboxylase (SAMDC) and transglutaminase (TGase), which were closely associated with the levels of free and conjugated PAs, were also detected. Results showed that under osmotic stress, the increases of the levels of non-covalently conjugated (non-CC) spermidine (Spd) and spermine (Spm), covalently conjugated (CC) putrescine (Put) and Spd in the plasma membrane of drought-tolerant Ganli No. 5 were more significant than those of drought-sensitive Suli No. 3, indicating that these conjugated PAs might be involved in the tolerance of plum seedlings to stress. Furthermore, the conjugated PAs were closely correlated with plum seedling growth, water retention capacity, plasma membrane damage degree, and hydrogen (H+)-ATPase activity in the plasma membrane. To get more complementary pieces of evidence, we subjected plum seedlings to combined treatments of PEG and exogenous PA (Spd and Spm), and an inhibitor of SAMDC [methylglyoxal-bis (guanylhydrazone), (MGBG)] or TGase (o-phenanthroline). These results collectively suggested that non-CC Spd and Spm, CC Put and Spd in plasma membrane might function in enhancing the tolerance of plum seedlings to osmotic stress by stabilizing membrane structure and therefore elevating H+-ATPase activity.

Persimmon Leaves (Diospyros kaki) Extract Enhances the Viability of Human Corneal Endothelial Cells by Improving Na+-K+-ATPase Activity

The Na+/K+-ATPase, present in the basolateral membrane of human corneal endothelial cells (HCECs), is known to play an important role for corneal transparency. Na+/K+-ATPase dysfunction is one of the major causes of corneal decompensation. The ethanol extract of Diospyros kaki (EEDK) has been reported to increase corneal cell viability. Thus, we treated HCECs with EEDK and studied its effects on HCECs survival and Na+/K+-ATPase against cytotoxic drugs like staurosporine (ST) and ouabain (OU). Firstly, survival assays, (MTT assay and live dead-imaging) showed that decreased HCECs viability by ST and OU was significantly recovered by EEDK co-treatment. Secondly, Na+/K+-ATPase activity assays revealed that EEDK enhanced Na+/K+-ATPase enzymatic activity (* p < 0.01) with/without ST and OU. Finally, Na+/K+-ATPase expression analysis (Western Blot and confocal microscopy) demonstrated that EEDK treatment with/without ST and OU facilitates Na+/K+-ATPase expression in HCECs. Taken together, our findings led us to the conclusion that EEDK might aid HCECs survival in vitro by increasing the activity and expression of Na+/K+-ATPase enzyme. Since Na+/K+-ATPase activity is important to maintain cellular function of HCECs, we suggest that EEDK can be a potential effective agent against corneal edema and related corneal disorders.

Valosin-containing protein regulates the stability of amyotrophic lateral sclerosis-causing fused in sarcoma granules in cells by changing ATP concentrations inside the granules

Fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) to form granules in cells, leading to pathogenic aggregations that cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Proteomics analysis revealed that FUS granules contain valosin-containing protein (VCP), a member of the AAA family ATPase. Confocal microscopy images showed that VCP co-localized in the FUS granules in cells. This study demonstrates that VCP in granules has a two-faced role in FUS granulation: VCP stabilizes de novo FUS granules, while VCP present in the granules for extended periods dissolves them. This VCP function relies on its ATPase activity to consume ATP in granules. VCP stabilizes de novo FUS by reducing intragranular ATP concentrations to a range below the cytosolic concentration. VCP continually consumes ATP during its stay in the granules, which eventually lowers ATP concentrations to a range that destabilizes the granules. VCP, therefore, acts as a timer to limit the residence of FUS granules in cells and thereby prohibits the FUS fibrillization that occurs in persistent granules. VCP ATPase activity plays a role in FUS granule turnover.

The Oxidative Status and Na +/K + -ATPase Activity in Obsessive-Compulsive Disorder: A Case Control Study

Background: Oxidative stress is involved in pathogenesis of some psychiatric disorders. To examine the role of oxidative stress in the etiopathogenesis of obsessive-compulsive disorder (OCD), we aimed to determine oxidative stress indices, including MDA levels in serum and red blood cells (RBC) membrane, total antioxidant capacity (TAC), serum glutathione (GSH) levels, serum antioxidant vitamins (A and E) and Na+/K+-ATPase activity in patients with the mentioned disorder vs. healthy controls.Method: 39 OCD patients diagnosed based on Diagnostic and Statistical Manual of Mental Disorders (DSM-V), and 39 volunteers’ healthy subjects were included in this study. MDA levels in serum and RBC membrane were measured using fluorimetric method. Serum TAC level, serum GSH level and Na+/K+-ATPase activity were also measured using spectrophotometric methods. Serum levels of vitamins were calculated by Reversed-phase high-performance liquid chromatography (RP-HPLC).Result: There was a significantly higher MDA level in serum (p<0.0001) and RBC membrane (p=0.002) of OCD patients compared with those in controls. A significant reduction in vitamin A (p=0.024) and vitamin E (p=0.001) levels was found in OCD patients vs. controls. There was significantly lower activity of erythrocyte membrane Na+-K+ ATPase in RBC membrane of OCD patients vs. controls (p<0.0001).Conclusion: Our findings indicate significantly higher levels MDA in both serum and RBC membrane, lower levels of serum vitamin A and E, and lower activity of membrane Na+-K+ ATPase in OCD patients compared to controls. These suggest an imbalance between oxidant and antioxidant factors in OCD patients that might play a fundamental role in the etiopathogenesis of OCD.

MIG-23 is involved in sperm migration in Ascaris suum

Sperm motility acquisition during maturation is essential for successful fertilization.Extracellular adenosine-5'-triphosphate (ATP) level mediation by MIG-23, which is a homolog of human ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), was required for major sperm protein filament dynamics and sperm motility in the nematode Ascaris suum. MIG-23 was localized on the sperm plasma membrane. During sperm activation, mitochondrial activity was increased dramatically, and a large amount of ATP was produced and stored in refringent granules (RGs). In addition, a portion of the produced ATP was released to the extracellular space through ATP channels, which were composed of innexins and localized on the sperm plasma membrane. Spermatozoa, instead of spermatids, hydrolyzed exogenous ATP and processed ecto-ATPase activity. MIG-23 contributed to the ecto-ATPase activity of spermatozoa. MIG-23 activity was interrupted, spermatozoa also decreased their ATP hydrolysis activity. Blocking MIG-23 activity resulted in an increase in the depolymerization rate of MSP filaments in pseudopodia, which eventually affected nematode sperm migration. Overall, our data imply that MIG-23, which contributes to the ecto-ATPase activity of spermatozoa, regulates sperm migration by modulating extracellular ATP levels.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.