SIRT3 aggravates metformin-induced energy stress and apoptosis in ovarian cancer cells.

Increasing evidence suggests that mitochondrial respiratory chain complex I participates in carcinogenesis and cancer progression by providing energy and maintaining mitochondrial function. However, the role of complex I in ovarian cancer is largely unknown. In this study we showed that metformin, considered to be an inhibitor of complex I, simultaneously inhibited cell growth and induced mitochondrial-related apoptosis in human ovarian cancer cells. Metformin interrupted cellular energy metabolism mainly by causing damage to complex I that impacted mitochondrial function. Additionally, treatment with metformin increased the activation of sirtuin 3 (SIRT3), a mitochondrial deacetylase. We demonstrated that SIRT3 overexpression aggravated metformin-induced apoptosis, energy stress and mitochondrial dysfunction. Moreover, treatment with metformin or SIRT3 overexpression increased activation of AMP-activated protein kinase (AMPK), a major sensor of cellular energy status. AMPK compensated for energy loss by increasing glycolysis. The impact of this was assessed by reducing glucose levels in the media or by using inhibitors (2-deoxyglucose, Compound C) of glycolysis and AMPK. The combination of these factors with metformin intensified cytotoxicity through further downregulation of ATP. Our study outlines an important role for SIRT3 in the antitumor effect of mitochondrial complex I inhibitors in human ovarian cancer cells. This effect appears to be mediated by induction of energy stress and apoptosis. Strategies that target the mitochondria could be enhanced by modulating glycolysis to further aggravate energy stress that may increase the antitumor effect.

Unconventional Luminescent Centers in Metastable Phases Created by Topochemical Reduction Reactions.

A low-temperature topochemical reduction strategy is used herein to prepare unconventional phosphors with luminescence covering the biological and/or telecommunications optical windows. This approach is demonstrated by using Bi(III)-doped Y2O3 (Y(2-x)Bi(x)O3) as a model system. Experimental results suggest that topochemical treatment of Y(2-x)Bi(x)O3 using CaH2 creates randomly distributed oxygen vacancies in the matrix, resulting in the change of the oxidation states of Bi to lower oxidation states. The change of the Bi coordination environments from the [BiO6] octahedra in Y(2-x)Bi(x)O3 to the oxygen-deficient [BiO(6-z)] polyhedra in reduced phases leads to a shift of the emission maximum from the visible to the near-infrared region. The generality of this approach was further demonstrated with other phosphors. Our findings suggest that this strategy can be used to explore Bi-doped or other classes of luminescent systems, thus opening up new avenues to develop novel optical materials.

Plasma levels of lysophosphatidic acid in ovarian cancer versus controls: a meta-analysis.

BACKGROUND: In this study, using a meta-analysis approach, we examined the correlation between serum levels of lysophosphastidic acid (LPA) and ovarian cancer (OC). METHODS: Relevant published studies were identified from multiple scientific literature databases by using a pre-determined electronic and manual search strategy. The search results were screened through a multi-step process to select high-quality case-control studies suitable for the present meta-analysis. Mean values and standardized mean differences (SMD) were calculated for plasma LPA levels. Two investigators independently extracted the data from the studies and performed data analysis using STATA software version 12.0 (Stata Corp, College Station, TX, USA). RESULTS: Nineteen case-control studies met our selection criteria and contained a total of 980 OC patients, 872 benign controls and 668 healthy controls. Our meta-analysis results revealed that the plasma levels of LPA in OC patients were significantly higher than benign controls (SMD = 2.36, 95% CI: 1.61-3.10, P < 0.001) and healthy controls (SMD = 2.32, 95% CI: 1.77-2.87, P < 0.001). Subgroup analysis by ethnicity showed that the plasma LPA levels in OC patients were significantly higher than the benign controls only in Asian populations (SMD = 2.52, 95% CI: 1.79-3.25, P < 0.001). However, a comparison between healthy controls and OC patients revealed that, in both Asians and Caucasians, the OC patients displayed significantly higher plasma LPA levels compared to healthy controls (all P < 0.05). CONCLUSION: Our meta-analysis showed strong evidence that a significantly higher plasma LPA levels are present in OC patients, compared to benign controls and healthy controls, and plasma LPA levels may be used as a biomarker or target of OC.

Suppression of chloride channel 3 expression facilitates sensitivity of human glioma U251 cells to cisplatin through concomitant inhibition of Akt and autophagy.

Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel-3 (ClC-3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3-II, beclin 1 and Atg12-Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC-3 by ClC-3 siRNA sensitized the apoptosis-resistant U251 cells to cisplatin-mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC-3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3-methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin-induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC-3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells.

The BH3 mimetic S1 induces autophagy through ER stress and disruption of Bcl-2/Beclin 1 interaction in human glioma U251 cells.

Previous results showed that a novel BH3 mimetic S1 could induce cell death in a wide range of cancer types in vitro through Bax/Bak-dependent apoptosis. We demonstrated that in addition to mitochondrial pathway apoptosis, endoplasmic reticulum (ER) stress-associated apoptosis was also induced by S1. Moreover, S1 can induce autophagy in U251 cells, which may occur through ER stress and disruption of the association of Bcl-2 and Beclin 1. Inhibition of autophagy by the autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ) increased S1-induced apoptosis. In conclusion, autophagy plays an important role in S1-induced U251 cell death.

Decreased purinergic inhibition of synaptic activity in a mouse model of Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a progressive neurodegenerative disorder characterized by accumulation of free cholesterol in lysosomes, mainly due to a mutation in the NPC1 gene. The pathophysiological basis of the neural disorders in NPC, however, is not well understood. We found that the hippocampal field excitatory postsynaptic potential (fEPSP) was enhanced in NPC1 mutant mice. A1-receptor antagonist or adenosine degrading enzyme enhanced the fEPSP in both types of mice, but had a much weaker effect in the mutant mice, suggesting less tonic inhibition of synaptic transmission by endogenous adenosine in the mutant. Further evidence showed impaired hippocampal long term potentiation (LTP) in mutant mice. Supplement of A1 agonist N6-Cyclopentyladenosine (CPA) partially rescued the impaired LTP in mutant mice. Moreover, adenosine release from hippocampal slices was significantly decreased in the mutant. The enhanced excitatory synaptic transmission and the decreased synaptic plasticity due to the decreased adenosine release in NPC brain may partially contribute to the neural disorders of NPC disease, such as seizures, neurodegeneration, and dementia.

Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism.

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass (ICM) that are able to self-renew or undergo differentiation depending on a complex interplay of extracellular signals and intracellular factors. However, the feedback regulation of differentiation-dependent ESC self-renewal is poorly understood. Retinoic acid (RA), a derivative of vitamin A, plays a critical role in ESC differentiation and embryogenesis. In the present study, we demonstrate that short-term treatment of murine (m) ESCs with RA during the early differentiation stage prevented spontaneous differentiation of mESCs. The RA-treated cells maintained self-renewal capacity and could differentiate into neuronal cells, cardiomyocytes, and visceral endoderm cells derived from three germ layers. The differentiation-inhibitory effect of RA was mimicked by conditioned medium from RA-treated ESCs and was accompanied with up-regulated expression of leukemia inhibitory factor (LIF), Wnt3a, Wnt5a, and Wnt6. Such RA-induced prevention of ESC differentiation was attenuated by a neutralizing antibody against LIF or by a specific Wnt antagonist Fz8-Fc and was totally reversed in the presence of both of them. Furthermore, knock-down of beta-catenin, a component of the Wnt signaling pathway, by small interfering RNA counteracted the effect of RA. In addition, RA treatment enhanced expression of endodermal markers GATA4 and AFP but inhibited expression of primitive ectodermal marker Fgf-5 and mesodermal marker Brachyury. These findings reveal a novel role of RA in ESC self-renewal and provide new insight into the regulatory mechanism of differentiation-dependent self-renewal of ESCs, in which Wnt proteins and LIF induced by RA have the synergistic action. The short-term treatment of ESCs with RA also offers a unique model system for study of the regulatory mechanism that controls self-renewal and specific germ-layer differentiation of ESCs.

Critical role of type 2 ryanodine receptor in mediating activity-dependent neurogenesis from embryonic stem cells.

Activity-induced neurogenesis via Ca(2+) entry may be important for establishing Hebbian neural network. However, it remains unclear whether intracellular Ca(2+) mobilization is required and which subtypes of Ca(2+) release channels expressed in Ca(2+) store organelles are involved in the activity-dependent neurogenesis. Here, we demonstrated that the activity of intracellular Ca(2+) signaling, expression of neuronal transcription factor NeuroD, and the rate of neurogenesis were significantly inhibited in neuronal cells derived from embryonic stem (ES) cells deficient in the Ca(2+) release channel type 2 ryanodine receptors (RyR2(-/-)). In wild-type (RyR2(+/+)) but not in RyR2(-/-) ES cells, activation of L-type Ca(2+) channels, GABA(A) receptors, or RyRs promoted neuronal differentiation, while inhibition of these channels/receptors had an opposite effect. Moreover, neuronal differentiation promoted by activation of GABA(A) receptors or L-type Ca(2+) channels in RyR2(+/+) cells was prevented by RyR inhibitors. No significant difference was detected in the expression level of GABA(A) receptors and L-type channels between neuronal cells derived from two types of ES cells. Thus, activity-induced Ca(2+) influx through L-type Ca(2+) channels alone is not sufficient in promoting neurogenesis. Instead, an intimate cooperation of L-type Ca(2+) channels with RyR2 is crucial for the activity-dependent neurogenesis induced by paracrine and/or autocrine GABA signaling.

Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells.

AIM: To investigate the developmental regulation of intracellular Ca2+ transients, an essential event in excitation-contraction coupling, during cardiomyocyte differentiation. METHODS: Using the embryonic stem (ES) cell in vitro differentiation system and pharmacological intervention, we investigated the molecular and functional regulation of Ca2+ handling proteins on the Ca2+ transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes (ESCM). RESULTS: Nifedipine, a selective antagonist of L-type Ca2+ channels, totally blocked Ca2+ transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. The Ca2+ transients of ESCM were also inhibited by both ryanodine [an inhibitor of ryanodine receptors (RyRs)] and 2-aminoethoxydipheylborate [2-APB, an inhibitor of inositol-1,4,5-trisphosphate receptors (IP3Rs)]. The inhibitory effect of ryanodine increased with the time of differentiation, while the effect of 2-APB decreased with the differentiation. Thapsigargin, an inhibitor of SR Ca2+-pump ATPase, inhibited Ca2+ transients equally at three differentiation stages that matched the expression profile. Na+ free solution, which inhibits Na+-Ca2+ exchanger (NCX) to extrude Ca2+ from cytosol, did not affect the amplitude of Ca2+ transients of ESCM until the latter differentiation stage, but it significantly enhanced the basal Ca2+ concentration. CONCLUSION: The Ca2+ transients in ESCM depend on both the sarcolemmal Ca2+ entry via L-type Ca2+ channels and the SR Ca2+ release from RyRs and IP3Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca2+ transients until the latter differentiation stage.

Comparison of protective effects between cultured Cordyceps militaris and natural Cordyceps sinensis against oxidative damage.

The Chinese herb DongChong-XiaCao originating from Cordyceps sinensis is widely used as a traditional medicine in China for treatment of a wide variety of diseases. The extracts of Cordyceps sinensis (CSE) and Cordyceps militaris (CME) are well-known for their biological effects. In the present study, the antioxidant efficiency of CME and CSE in protecting lipid, protein, and low-density lipoprotein (LDL) against oxidative damage was investigated. CME and CSE showed weakly inhibitory effect on liposome oxidation, that of CME being superior to that of CSE. As for the protein oxidation model system, the inhibitory effect of CME on protein oxidation was inferior to that of CSE. CME and CSE at 1.0 mg/mL showed 50.5 and 67.1% inhibition of LDL oxidation, respectively. The contents of bioactive ingredients cordycepin and adenosine in CME are higher than those of CSE; however, both cordycepin and adenosine showed no significant antioxidant activity as determined by the Trolox equivalent antioxidant capacity method. Polyphenolic and flavonoid contents are 60.2 and 0.598 microg/mL in CME and 31.8 and 0.616 microg/mL in CSE, respectively, which may in part be responsible for their antioxidant activities. In addition, a polysaccharide present in CME and CSE displayed antioxidant activity, which suggested that the activity might be derived partly from polysaccharides of CME and CSE. The tendency to scavenge the ABTS(*)(+) free radical and the reducing ability of CME and CSE display concentration-dependent manners, suggesting that CME and CSE may be potent hydrogen donators. On the basis of the results obtained, the protective effects of CME and CSE against oxidative damage of biomolecules are a result of their free radical scavenging abilities.

Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells.

In adult myocardium, excitation-contraction coupling is critically regulated by sarcoplasmic reticulum (SR) Ca2+ release via type 2 ryanodine receptor (RyR2), but generally, it is believed that SR-function is rudimentary in the fetal heart and in embryonic stem (ES) cell-derived cardiomyocytes (ESCMs), a possible source for cell replacement therapies. This study used wild-type (RyR2+/+) and RyR2 null (RyR2-/-) ESCMs as an in vitro model of cardiomyogenesis, together with pharmacological approaches and expression profiles of genes relevant for SR function, to elucidate the functional importance of RyR2 and SR on the regulation of Ca2+ transients and contraction during early cardiomyocyte development. During differentiation of RyR2+/+ ESCMs, SR function developed progressively with increased basal cytosolic free Ca2+ concentration ([Ca2+]i), enhanced frequency and amplitude, and decreased duration of Ca2+ transients that were inhibited by ryanodine and thapsigargin. These functional traits correlated with SR Ca2+ load and the expression of RyR2, SERCA2a, and phospholamban. RyR2-/- ESCMs, comparatively, demonstrated a significantly prolonged time-to-peak and reduced frequency of Ca2+ transients and contractions. Beta-adrenergic stimulation of RyR2+/+ ESCMs increased the frequency and amplitude of Ca2+ transients with differentiation but was much weaker in RyR2-/- ESCMs. We conclude that functional SR and control of RyR2-mediated SR Ca2+ release directly contribute to the spontaneous and beta-adrenergic receptor-stimulated contraction of ESCMs, even at very immature stages of development.