Relationship between delayed luminescence emission and mitochondrial status in Saccharomyces cerevisiae

Delayed luminescence (DL) is gradually used in various detection of biological systems as a rapid detection technique, however, its biological mechanism was still not clear. In this study, a new model of DL detection system for liquid biological samples is established to investigate the DL emission of Saccharomyces cerevisiae cells cultured in different glucose concentrations. We analyzed the relationship between the DL emission and cell growth, cell vitality, mitochondrial morphology, mitochondrial DNA (mtDNA) copy number, adenosine triphosphate (ATP), oxygen consumption rate (OCR), as well as mitochondria membrane potential (MMP) in S. cerevisiae cells cultured with 0.01, 0.05, 0.15, 3, 10 and 20 g/L glucose respectively. It was found that the DL emission had strong correlation with mitochondrial morphology, OCR, and MMP. The results suggested that DL is an indicator of mitochondria status under different glucose supply conditions, and may be an effective method to detect mitochondrial metabolism related disorders.

GRPEL2 Knockdown Exerts Redox Regulation in Glioblastoma

Malignant brain tumors are responsible for catastrophic morbidity and mortality globally. Among them, glioblastoma multiforme (GBM) bears the worst prognosis. The GrpE-like 2 homolog (GRPEL2) plays a crucial role in regulating mitochondrial protein import and redox homeostasis. However, the role of GRPEL2 in human glioblastoma has yet to be clarified. In this study, we investigated the function of GRPEL2 in glioma. Based on bioinformatics analyses from the Cancer Gene Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), we inferred that GRPEL2 expression positively correlates with WHO tumor grade (p < 0.001), IDH mutation status (p < 0.001), oligodendroglial differentiation (p < 0.001), and overall survival (p < 0.001) in glioma datasets. Functional validation in LN229 and GBM8401 GBM cells showed that GRPEL2 knockdown efficiently inhibited cellular proliferation. Moreover, GRPEL2 suppression induced cell cycle arrest at the sub-G1 phase. Furthermore, GRPEL2 silencing decreased intracellular reactive oxygen species (ROS) without impending mitochondria membrane potential. The cellular oxidative respiration measured with a Seahorse XFp analyzer exhibited a reduction of the oxygen consumption rate (OCR) in GBM cells by siGRPEL2, which subsequently enhanced autophagy and senescence in glioblastoma cells. Taken together, GRPEL2 is a novel redox regulator of mitochondria bioenergetics and a potential target for treating GBM in the future.

A conserved expression signature predicts growth rate and reveals cell & lineage-specific differences

Isogenic cells cultured together show heterogeneity in their proliferation rate. To determine the differences between fast and slow-proliferating cells, we developed a method to sort cells by proliferation rate, and performed RNA-seq on slow and fast proliferating subpopulations of pluripotent mouse embryonic stem cells (mESCs) and mouse fibroblasts. We found that slowly proliferating mESCs have a more naïve pluripotent character. We identified an evolutionarily conserved proliferation-correlated transcriptomic signature that is common to all eukaryotes: fast cells have higher expression of genes for protein synthesis and protein degradation. This signature accurately predicted growth rate in yeast and cancer cells, and identified lineage-specific proliferation dynamics during development, using C. elegans scRNA-seq data. In contrast, sorting by mitochondria membrane potential revealed a highly cell-type specific mitochondria-state related transcriptome. mESCs with hyperpolarized mitochondria are fast proliferating, while the opposite is true for fibroblasts. The mitochondrial electron transport chain inhibitor antimycin affected slow and fast subpopulations differently. While a major transcriptional-signature associated with cell-to-cell heterogeneity in proliferation is conserved, the metabolic and energetic dependency of cell proliferation is cell-type specific.

Actinomycin V Induces Apoptosis Associated with Mitochondrial and PI3K/AKT Pathways in Human CRC Cells

Actinomycin (Act) V, an analogue of Act D, presented stronger antitumor activity and less hepatorenal toxicity than Act D in our previous studies, which is worthy of further investigation. We hereby report that Act V induces apoptosis via mitochondrial and PI3K/AKT pathways in colorectal cancer (CRC) cells. Act V-induced apoptosis was characterized by mitochondrial dysfunction, with loss of mitochondria membrane potential (MMP) and cytochrome c release, which then activated cleaved caspase-9, cleaved caspase-3, and cleaved PARP, revealing that it was related to the mitochondrial pathway, and the apoptotic trendency can be reversed by caspase inhibitor Z-VAD-FMK. Furthermore, we proved that Act V significantly inhibited PI3K/AKT signalling in HCT-116 cells using cell experiments in vitro, and it also presented a potential targeted PI3Kα inhibition using computer docking models. Further elucidation revealed that it exhibited a 28-fold greater potency than the PI3K inhibitor LY294002 on PI3K inhibition efficacy. Taken together, Act V, as a superior potential replacement of Act D, is a potential candidate for inhibiting the PI3K/AKT pathway and is worthy of more pre-clinical studies in the therapy of CRC.

Palmitate Enhances The Efficacy of Cisplatin And Doxorubicin Against Human Endometrial Carcinoma Cells

Background: Endometrial cancer is the most common gynecological cancer worldwide. Overexpression of fatty acid synthase is a common molecular feature of a subgroup of sex steroid-related cancers associated with poor prognoses, including endometrial cancers. The saturated fatty acid palmitate reportedly induces lipotoxicity and cell death by inducing oxidative stress in many cell types. Here, we explored the effects of palmitate combined with doxorubicin or cisplatin in the HEC-1-A and RL95-2 human endometrial cancer cell lines. Methods: Endometrial cancer cells were cultured with in vitro and treated with palmitate, doxorubicin, and cisplatin. Cell metabolic activity and combination index was measured using MTT assay. Protein expression was assessed with western blotting. Flow cytometry was used to examine the cell cycle profiles, cell proliferation, apoptosis, ROS, mitochondria membrane potential, and mitochondrial mass. Immunocytochemistry was used to investigate the mitochondrial morphology.Results: Physiological concentrations of exogenous palmitate significantly increased cell cycle arrest, DNA damage, autophagy and apoptosis in both RL95-2 and HEC-1-A cells. It also increased the chemosensitivity of both cell types. Notably, we did not observe that palmitate lipotoxicity reflected increased levels of reactive oxygen species, suggesting palmitate acts via a different mechanism in endometrial cancer.Conclusion: This study provides a potential therapeutic strategy in which palmitate is used as an adjuvant in the treatment of endometrial cancer.

Oridonin enhances antitumor effects of doxorubicin in human osteosarcoma cells

Background Doxorubicin is the chemotherapeutic drug of choice in osteosarcoma treatment, but its cumulative administration causes dilated cardiomyopathy. Combination therapy represents a potential strategy to reduce the therapeutic dosage of the chemotherapeutic agent and minimize its side effects. The aim of this study was to evaluate the potential of oridonin, a natural product from the medicinal herb Rabdosia rubescens, to act in combination with doxorubicin for osteosarcoma treatment. To date, there are no reports of the simultaneous administration of both drugs in osteosarcoma therapy. Methods The combined administration of different doses of oridonin and doxorubicin, as compared with the drugs alone, were tested in an in vitro model of osteosarcoma. The synergistic effect of the drugs on cell death was assessed by alamarBlue™ and by CompuSyn software. Early and late apoptosis markers (JC-1 fluorescence and Annexin V immunofluorescence), as well as the production of reactive oxygen species, were evaluated by flow cytometry. Western blot was used to assess the expression of anti-apoptotic proteins. Results Oridonin and doxorubicin presented a synergistic cytotoxic effect in osteosarcoma cells. In the presence of sub-cytotoxic concentrations of the natural product, there was an increased accumulation of intracellular doxorubicin, increased levels of reactive oxygen species (ROS), alteration of mitochondria membrane potential and a higher rate of apoptosis. Conclusion The combined use of oridonin and doxorubicin could help to reduce the clinical dosage of doxorubicin and its dangerous side effects.

Rice Husk Silica Liquid Protects Pancreatic β Cells from Streptozotocin-Induced Oxidative Damage

Type 2 diabetes mellitus is a complex multifactorial disease characterized by insulin resistance and dysfunction of pancreatic β-cells. Rice husk silica liquid (RHSL) is derived from rice husks and has not been explored in diabetes mellitus until now. Previous studies showed that rice husk is enriched with silica, and its silica nanoparticles are higher more biocompatible. To investigate the potential protective role of RHSL on pancreatic β cells, we utilized RIN-m5F pancreatic β cells and explored RHSL effect after streptozotocin (STZ)-stimulation. The recovery effects of RHSL were evaluated using flow cytometry, Western blotting, and immunofluorescence analysis. Results of our study showed that RHSL reversed the cell viability, insulin secretion, reactive oxygen species (ROS) production, and the change of mitochondria membrane potential (ΔΨm) in STZ-treated RIN-m5F cells. Moreover, the expression of phospho-receptor-interacting protein 3 (p-RIP3) and cleaved-poly (ADP-ribose) polymerase (PARP), phospho-mammalian target of rapamycin (p-mTOR), and sequestosome-1 (p62/SQSTM1) were significantly decreased, while the transition of light chain (LC)3-I to LC3-II was markedly increased after RHSL treatment in STZ-induced RIN-m5F cells. Interestingly, using autophagy inhibitors such as 3-methyladenine (3-MA) and chloroquine (CQ) both showed an increase in cleaved-PARP protein level, indicating apoptosis induction. Taken together, this study demonstrated that RHSL induced autophagy and alleviated STZ-induced ROS-mediated apoptosis in RIN-m5F cells.

Enzymatic Degradation of Gracilariopsis lemaneiformis Polysaccharide and the Antioxidant Activity of Its Degradation Products

Gracilariopsis lemaneiformis polysaccharides (GLP) were degraded using pectinase, glucoamylase, cellulase, xylanase, and β-dextranase into low-molecular-weight polysaccharides, namely, GPP, GGP, GCP, GXP, and GDP, respectively, and their antioxidant capacities were investigated. The degraded GLPs showed higher antioxidant activities than natural GLP, and GDP exhibited the highest antioxidant activity. After the optimization of degradation conditions through single-factor and orthogonal optimization experiments, four polysaccharide fractions (GDP1, GDP2, GDP3, and GDP4) with high antioxidant abilities (hydroxyl radical scavenging activity, DPPH radical scavenging activity, reduction capacity, and total antioxidant capacity) were obtained. Their cytoprotective activities against H2O2-induced oxidative damage in human fetal lung fibroblast 1 (HFL1) cells were examined. Results suggested that GDP pretreatment can significantly improve cell viability, reduce reactive oxygen species and malonaldehyde levels, improve antioxidant enzyme activity and mitochondria membrane potential, and alleviate oxidative damage in HFL1 cells. Thus, the enzyme degradation of GLP with β-dextranase can significantly improve its antioxidant activity, and GDP might be a suitable source of natural antioxidants.

The Potential Use of Sialic Acid From Edible Bird’s Nest to Attenuate Mitochondrial Dysfunction by In Vitro Study

Edible bird’s nest (EBN) is one of the expensive functional foods in herbal medicine. One of the major glyconutrients in EBN is sialic acid, which has a beneficial effect on neurological and intellectual capability in mammals. The aims of this research were to study the effects of sialic acid from EBN on cell viability and to determine its effect on mitochondria membrane potential (MtMP) in Caco-2, SK-N-MC, SH-SY5Y, and PC-12 cell lines. Fourteen samples of raw EBN were collected from four different states in Malaysia. The confluency of the epithelial monolayers measurement of the tight junction for all the cell lines was determined using transepithelial electrical resistance (TEER), and the sialic acid uptake study in cell lines was determined by using ultra-high performance liquid chromatography (UHPLC). The MTT assay was conducted for cell viability study. The MtMP in cell lines was determined using the Mito Probe JC-1 Assay by flow cytometer analysis. We have recorded a statistically significant difference between the uptake of sialic acid from EBN and the standard solution. A higher amount of sialic acid was absorbed by the cells from extract of EBN compared to the standard solution. The amounts of sialic acid uptake in Caco-2, SK-N-MC, SH-SY5Y, and PC-12 cell lines were (0.019 ± 0.001), (0.034 ± 0.006), (0.021 ± 0.002), and (0.025 ± 0.000) µmol/L, respectively. The MTT results indicated that the concentration of sialic acid increased the cell viability and showed no cytotoxicity effects on cell lines when they were exposed to the sialic acid extract and sialic acid standard at all the tested concentrations. The number of active mitochondria was found to be significantly higher in SH-SY5Y cell lines with a 195% increase when treated with sialic acid from EBN. Although many researchers around the globe use SH-SY5Y and SK-N-MC for Alzheimer’s disease (AD) study, based on our finding, SH-SY5Y was found to be the most suitable cell line for AD study by in vitro works where it has a known relationship with mitochondrial dysfunction.