Daidzein Synergizes with Gefitinib to Induce ROS/JNK/c-Jun Activation and Inhibit EGFR-STAT/AKT/ERK Pathways to enhance Lung Adenocarcinoma cells chemosensitivity

Lung cancer is the major cause of cancer associated mortality. Mutations in EGFR have been implicated in lung cancer pathogenesis. Gefitinib (GF) is a RTKI (receptor tyrosine kinase inhibitor) first-choice drug for EGFR mutated advanced lung cancer. However, drug toxicity and cancer cell resistance lead to treatment failure. Consequently, new therapeutic strategies are urgently required. Therefore, this study was aimed at identifying tumor suppressive compounds that can synergistically improve Gefitinib chemosensitivity in the lung cancer treatment. Medicinal plants offer a vast platform for the development of novel anticancer agents. Daidzein (DZ) is an isoflavone compound extracted from soy plants and has been shown to possess many medicinal benefits. The anticancer potential of GF and DZ combination treatment was investigated using MTT, western blot, fluorescent microscopy imaging, flow cytometry and nude mice tumor xenograft techniques. Our results demonstrate that DZ synergistically induces c-Jun nuclear translocation through ROS/ASK1/JNK and downregulates EGFR-STAT/AKT/ERK pathways to activate apoptosis and a G0/G1 phase cell cycle blockade. In in-vivo, the combination treatment significantly suppressed A549 lung cancer cells tumor xenograft growth without noticeable toxicity. Daidzein supplements with current chemotherapeutic agents may well be an alternative strategy to improve the treatment efficacy of lung adenocarcinoma.     

Berberine Protects against Neuronal Damage via Suppression of Glia-Mediated Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) triggers a series of neuroinflammatory processes that contribute to evolution of neuronal injury. The present study investigated the neuroprotective effects and anti-inflammatory actions of berberine, an isoquinoline alkaloid, in both in vitro and in vivo TBI models. Mice subjected to controlled cortical impact injury were injected with berberine (10 mg·kg⁻¹) or vehicle 10 min after injury. In addition to behavioral studies and histology analysis, blood-brain barrier (BBB) permeability and brain water content were determined. Expression of PI3K/Akt and Erk signaling and inflammatory mediators were also analyzed. The protective effect of berberine was also investigated in cultured neurons either subjected to stretch injury or exposed to conditioned media with activated microglia. Berberine significantly attenuated functional deficits and brain damage associated with TBI up to day 28 post-injury. Berberine also reduced neuronal death, apoptosis, BBB permeability, and brain edema at day 1 post-injury. These changes coincided with a marked reduction in leukocyte infiltration, microglial activation, matrix metalloproteinase-9 activity, and expression of inflammatory mediators. Berberine had no effect on Akt or Erk 1/2 phosphorylation. In mixed glial cultures, berberine reduced TLR4/MyD88/NF-κB signaling. Berberine also attenuated neuronal death induced by microglial conditioned media; however, it did not directly protect cultured neurons subjected to stretch injury. Moreover, administration of berberine at 3 h post-injury also reduced TBI-induced neuronal damage, apoptosis and inflammation in vivo. Berberine reduces TBI-induced brain damage by limiting the production of inflammatory mediators by glial cells, rather than by a direct neuroprotective effect.     

Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle.

The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation.     

GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress

Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634∼736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H₂O₂-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H₂O₂ insult.     

Electrical stimulation promotes nerve growth factor-induced neurite outgrowth and signaling

Background

Neurotrophins are important regulators for neural development and regeneration. Nerve growth factor (NGF) therapy has been tested in various models of neural injury and degeneration. However, whether NGF can reach target tissues and maintain effective concentration for a certain period of time remains uncertain. To facilitate neural regeneration, we investigate the possibility of combining NGF and electrical stimulation (ES) in promoting neurite outgrowth, an essential process during neural regeneration.

Methods

PC12 cells were seeded on collagen and indium tin oxide (ITO)-coated area on the transparent conductive devices. Cells were then subjected to the combination of ES and NGF treatment. Neurite outgrowth was compared.

Results

Our findings suggest that ES of 100 mV/mm together with NGF provides optimal effect on neurite outgrowth of PC12 cells. ES increases NGF-induced neurite length but reduces neurite branching, indicative of its primary effect on neurite elongation instead of initiation. One mechanism that ES enhances neurite outgrowth is through increasing NGF-induced phosphorylation of ERK1/2 (pERK1/2) and expression of Egr1 gene. ES has previously been demonstrated to increase the activity of protein kinase C (PKC). Our result indicates that activating PKC further increases NGF-induced pERK1/2 and thus neurite outgrowth.

Conclusion

It is likely that ES promotes NGF-induced neurite outgrowth through modulating the activity of ERK1/2.

General significance

Findings from this study suggest that combining ES and NGF provides a promising strategy for promoting neurite outgrowth.     

RANKL increases migration of human lung cancer cells through intercellular adhesion molecule-1 up-regulation

Invasion of distant tissues by tumor cells is the primary cause of therapeutic failure in the treatment of malignant lung cancer cells. Receptor activator of nuclear factor-κB ligand (RANKL) and its receptor, RANK, play a key role in osteoclastogenesis and tumor metastasis. Intercellular adhesion molecule-1 (ICAM-1, also called CD54), a member of the immunoglobulin supergene family, is an inducible surface glycoprotein that mediates adhesion-dependent cell-to-cell interactions. The effects of RANKL on cell migration and ICAM-1 expression in human lung cancer cells are largely unknown. We found that RANKL directed the migration and increased ICAM-1 expression in human lung cancer (A549) cells. Pretreatment of A549 cells with the MAPK kinase (MEK) inhibitor PD98059 or U0126 inhibited RANKL-mediated migration and ICAM-1 expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, an NF-κB inhibitor (PDTC) and IκB protease inhibitor (TPCK) also inhibited RANKL-mediated cell migration and ICAM-1 up-regulation. Taken together, these results suggest that the RANKL and RANK interaction acts through MEK/ERK, which in turn activates NF-κB, resulting in the activation of ICAM-1 and contributing to the migration of human lung cancer cells.     

AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1

We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A–mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.     

LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

Effect of a two-month detraining on glucose tolerance and insulin sensitivity in athletes--link to adrenal steroid hormones

Reduction in physical activity has been demonstrated to associate with the increased risk in insulin resistance and type 2 diabetes. To determine whether alteration in insulinemia, due to abstention from regular exercise training, is associated with changes in serum dehydroepiandrosterone sulfate (DHEA-S) and cortisol, 18 highly trained badminton players (21.2 +/- 0.3 years) were enrolled into a 2-month detraining study. Fasting serum insulin, glucose, DHEA-S, and cortisol were determined at trained state and at day 60 of detraining. Glucose tolerance and insulin sensitivity were assessed by an oral glucose tolerance test (OGTT). The 2-month detraining increased fasting glucose and insulin concentrations and body weight slightly, but did not significantly affect glucose tolerance and insulin response curve, in which 10 subjects had increased and 8 subjects had slightly decreased in the area under curve for insulin (IAUC). In the subjects with increased IAUC, serum cortisol was also elevated (from 0.44 +/- 0.07 to 0.83 +/- 0.26 U/l, P < 0.05) in parallel, and serum creatine kinase (CK) was unaltered during detraining. Whereas in the subjects with decreased IAUC, serum cortisol (from 0.51 +/- 0.19 to 0.54 +/- 0.14 U/l, no significance) was not changed and serum creatine kinase (from 461 +/- 179 to 151 +/- 21 U/l) was decreased during detraining. Two groups of detrained subjects exhibited a similar reduction in serum DHEA-S levels and slight elevation in body weight. The novel finding of the study is that the changes in serum cortisol, but not DHEA-S, were associated with the change in insulin sensitivity during early phase of lifestyle change from physically active to sedentary, and this response appears to be varied individually among athletes.     

Biochemical and biological characterization of a neuroendocrine-associated phosphatase

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling.