Interferon-β Produces Synergistic Combinatory Anti-Tumor Effects with Cisplatin or Pemetrexed on Mesothelioma Cells

Interferons (IFNs) have been tested for the therapeutic effects in various types of malignancy, but mechanisms of the anti-tumors effects and the differential biological activities among IFN members are dependent on respective cell types. In this study, we examined growth inhibitory activities of type I and III IFNs on 5 kinds of human mesothelioma cells bearing wild-type p53 gene, and showed that type I IFNs but not type III IFNs decreased the cell viabilities. Moreover, growth inhibitory activities and up-regulated expression levels of the major histocompatibility complexes class I antigens were greater with IFN-β than with IFN-α treatments. Cell cycle analyses demonstrated that type I IFNs increased S- and G2/M-phase populations, and subsequently sub-G1-phase fractions. The cell cycle changes were also greater with IFN-β than IFN-α treatments, and these data collectively showed that IFN-β had stronger biological activities than IFN-α in mesothelioma. Type I IFNs-treated cells increased p53 expression and the phosphorylation levels, and activated apoptotic pathways. A combinatory use of IFN-β and cisplatin or pemetrexed, both of which are the current first-line chemotherapeutic agents for mesothelioma, produced synergistic anti-tumor effects, which were also evidenced by increased sub-G1-phase fractions. These data demonstrated firstly to our knowledge that IFN-β produced synergistic anti-tumor effects with cisplatin or pemetrexed on mesothelioma through up-regulated p53 expression.     

The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.     

Downregulation of the small GTPase Ras-related nuclear protein accelerates cellular ageing

Background

The small GTPase Ran, Ras-related nuclear protein, plays important roles in multiple fundamental cellular functions such as nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, by binding to either GTP or GDP as a molecular switch. Although it has been clinically demonstrated that Ran is highly expressed in multiple types of cancer cells and specimens, the physiological significance of Ran expression levels is unknown.

Methods

During the long-term culture of normal mammalian cells, we found that the endogenous Ran level gradually reduced in a passage-dependent manner. To examine the physiological significance of Ran reduction, we first performed small interfering RNA (siRNA)-mediated abrogation of Ran in human diploid fibroblasts.

Results

Ran-depleted cells showed several senescent phenotypes. Furthermore, we found that nuclear accumulation of importin α, which was also observed in cells treated with siRNA against CAS, a specific export factor for importin α, occurred in the Ran-depleted cells before the cells showed senescent phenotypes. Further, the CAS-depleted cells also exhibited cellular senescence. Indeed, importin α showed predominant nuclear localisation in a passage-dependent manner.

Conclusions

Reduction in Ran levels causes cytoplasmic decrease and nuclear accumulation of importin α leading to cellular senescence in normal cells.

General significance

The amount of intracellular Ran may be critically related to cell fate determination, such as malignant transformation and senescence. The cellular ageing process may proceed through gradual regression of Ran-dependent nucleocytoplasmic transport competency.     

Stimulation of σ1-receptor restores abnormal mitochondrial Ca mobilization and ATP production following cardiac hypertrophy

Background

We previously reported that the σ₁-receptor (σ₁R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ₁R stimulation with the selective σ₁R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ₁R localized in the sarcoplasmic reticulum (SR).

Methods

We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ₁R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ₁R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).

Results

σ₁R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca^2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca^2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.

Conclusions

σ₁R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca^2 + mobilization and ATP production via σ₁R stimulation.

General significance

Our observations suggest that σ₁R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.     

Controlled accumulation of vitellin in Bombyx eggs

Summary

Implantation of female fat body and ovary discs into the young male pupae brought about vitellin accumulation in the mature eggs developed in male hosts. The amounts of vitellin increased according to the increasing amounts of implanted fat body, and the vitellin synthesis activity of female fat body in male hosts was similar to that found in female hosts. When implanted into male pupae, larval fat body having no ability to produce vitellogenin in situ could bring about vitellin accumulation in the eggs. No accumulation of vitellin was induced by implantation of male fat body.     

Oxidative stress is associated with aluminum toxicity recovery in apex of pea root

Aims

Although many studies on the mechanism of Al toxicity and tolerance have been conducted independently, events occurring during the recovery process from Al injury is limited. This study was to investigate Al toxicity recovery mechanism focusing in morphological and physiological aspect.

Methods

We investigated the mechanisms underlying Al toxicity recovery in terms of oxidative stress using the pea root apex as a model system.

Results

The accumulation of reactive oxygen species was remarkably high in the root under continued Al treatment but decreased in the recovering root. The superoxide anion exuded in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) showed a similar tendency with respect to the accumulation of reactive oxygen species. A similar pattern of lignin content and superoxide dismutase activity was observed among the treatments, while the increased peroxidation in the root under continued Al treatment did not decline with recovery treatment. A longitudinal section of the root under continued Al treatment showed the accumulation of superoxide anion, lignin and peroxide (H₂O₂) at the epidermal and outer cortex region where the Al induced injuries, including ruptures, are detected.

Conclusions

Oxidative stress is associated with the mechanism of Al toxicity recovery. The recovery process might include the elongation of the central cylinder as a consequence of the oxidative stress-induced formation of the zonal region (ZR). The results further suggest a plausible role for the ZR in the programmed cell death-like function involved in Al toxicity recovery.