Inhibitory effects of Dulcitol on rat C6 glioma by regulating autophagy pathway

Abstract

In the study, we treated C6 rat glioma cells with 25?mg/ml Dulcitol for 24?h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to detect cellular growth. The measurements of the superoxide dismutase (SOD), malondialdehyde (MDA) and catalase (CAT) were used to assess oxidative stress level. Western was performed to detect the autophagy and apoptosis expression. The data showed that Dulcitol significantly decreased the cell viability, upregulated the Bax level in mitochondria and the Cytochrome C level in cytoplasm, and downregulated anti-apoptotic protein Bcl-xl. Moreover, it enhanced MDA level, reduced CAT and SOD activities, decreased LC3-II/LC3-I ratio, and increased P62 expression. However, rapamycin increased autophagy level and cell viability, and decreased ROS in Dulcitol treated C6 cells. Moreover, Dulcitol inhibited the glioma growth and enhanced survival in vivo. These results suggest that Dulcitol evidently increase cellular ROS levels and apoptosis in glioma cells, which can be significantly regulated by autophagy.     

Ursolic Acid Enhances the Sensitivity of MCF-7 and MDA-MB-231 Cells to Epirubicin by Modulating the Autophagy Pathway

Breast cancer is the leading cause of cancer death among women in the world, and its morbidity and mortality are increasing year by year. Epirubicin (EPI) is a commonly used drug for the treatment of breast cancer but unfortunately can cause cardiac toxicity in patients because of dose accumulation. Therefore, there is an urgent need for new therapies to enhance the sensitivity of breast cancer cells to EPI. In this study, we found ursolic acid (UA) can significantly improve the drug sensitivity of human breast cancer MCF-7/MDA-MB-231 cells to EPI. Next, we observed that the co-treatment of UA and EPI can up-regulate the expression of autophagy-related proteins Beclin-1, LC3-II/LC3-I, Atg5, and Atg7, and decrease the expression levels of PI3K and AKT, which indicates that the potential mechanism should be carried out by the regulating class III PI3K(VPS34)/Beclin-1 pathway and PI3K/AKT/mTOR pathway. Furthermore, we found the autophagy inhibitor 3-methyladenine (3-MA) could significantly reverse the inhibitory effect of co-treatment of UA and EPI on MCF-7 and MDA-MB-231 cells. These findings indicate that UA can dramatically enhance the sensitivity of MCF-7 and MDA-MB-231 cells to EPI by modulating the autophagy pathway. Our study may provide a new therapeutic strategy for combination therapy.     

Cephalotaxine Inhibits the Survival of Leukemia Cells by Activating Mitochondrial Apoptosis Pathway and Inhibiting Autophagy Flow

Cephalotaxine (CET) is a natural alkaloid with potent antileukemia effects. However, its underlying molecular mechanism has not been well understood. In this study, we verified that CET significantly inhibited the viability of various leukemia cells, including HL-60, NB4, Jurkat, K562, Raji and MOLT-4. RNA-sequencing and bioinformatics analysis revealed that CET causes mitochondrial function change. Mechanism research indicated that CET activated the mitochondrial apoptosis pathway by reducing the mitochondrial membrane potential, downregulating anti-apoptotic Bcl-2 protein and upregulating pro-apoptotic Bak protein. In addition, the autophagy signaling pathway was highly enriched by RNA-seq analysis. Then, we found that CET blocked the fluorescence colocation of MitoTracker Green and LysoTracker Red and upregulated the level of LC3-II and p62, which indicated that autophagy flow was impaired. Further results demonstrated that CET could impair lysosomal acidification and block autophagy flow. Finally, inhibiting autophagy flow could aggravate apoptosis of HL-60 cells induced by CET. In summary, this study demonstrated that CET exerted antileukemia effects through activation of the mitochondria-dependent pathway and by impairing autophagy flow. Our research provides new insights into the molecular mechanisms of CET in the treatment of leukemia.     

Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.     

Saponins Extracted from Tea (Camellia Sinensis) Flowers Induces Autophagy in Ovarian Cancer Cells

Tea flower saponins (TFS) possess effective anticancer properties. The diversity and complexity of TFS increases the difficulty of their extraction and purification from tea flowers. Here, multiple methods including solvent extraction, microporous resin separation and preparative HPLC separation were used to obtain TFS with a yield of 0.34%. Furthermore, we revealed that TFS induced autophagy—as evidenced by an increase in MDC-positive cell populations and mCherry-LC3B-labeled autolysosomes and an upregulation of LC3II protein levels. 3-MA reversed the decrease in cell viability induced by TFS, showing that TFS induced autophagic cell death. TFS-induced autophagy was not dependent on the Akt/mTOR/p70S6K signaling pathway. TFS-induced autophagy in OVCAR-3 cells was accompanied by ERK pathway activation and reactive oxygen species (ROS) generation. This paper is the first report of TFS-mediated autophagy of ovarian cancer cells. These results provide new insights for future studies of the anti-cancer effects of TFS.     

Autophagic Activation and Decrease of Plasma Membrane Cholesterol Contribute to Anticancer Activities in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC), an aggressive subtype of pulmonary carcinomas with high mortality, accounts for 85% of all lung cancers. Drug resistance and high recurrence rates impede the chemotherapeutic effect, making it urgent to develop new anti-NSCLC agents. Recently, we have demonstrated that para-toluenesulfonamide is a potential anti-tumor agent in human castration-resistant prostate cancer (CRPC) through inhibition of Akt/mTOR/p70S6 kinase pathway and lipid raft disruption. In the current study, we further addressed the critical role of cholesterol-enriched membrane microdomain and autophagic activation to para-toluenesulfonamide action in killing NSCLC. Similar in CRPC, para-toluenesulfonamide inhibited the Akt/mTOR/p70S6K pathway in NSCLC cell lines NCI-H460 and A549, leading to G1 arrest of the cell cycle and apoptosis. Para-toluenesulfonamide significantly decreased the cholesterol levels of plasma membrane. External cholesterol supplement rescued para-toluenesulfonamide-mediated effects. Para-toluenesulfonamide induced a profound increase of LC3-II protein expression and a significant decrease of p62 expression. Double staining of lysosomes and cellular cholesterol showed para-toluenesulfonamide-induced lysosomal transportation of cholesterol, which was validated using flow cytometric analysis of lysosome staining. Moreover, autophagy inhibitors could blunt para-toluenesulfonamide-induced effect, indicating autophagy induction. In conclusion, the data suggest that para-toluenesulfonamide is an effective anticancer agent against NSCLC through G1 checkpoint arrest and apoptotic cell death. The disturbance of membrane cholesterol levels and autophagic activation may play a crucial role to para-toluenesulfonamide action.     

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.     

A Quassinoid Diterpenoid Eurycomanone from Eurycoma longifolia Jack Exerts Anti-Cancer Effect through Autophagy Inhibition

Eurycomanone (EN) is one of the representative quassinoid diterpenoids from roots of Eurycoma longifolia Jack, a natural medicine that is widely distributed in Southeast Asia. Previous studies showed that EN induces cancer cell apoptosis and exhibits anti-cancer activity, but the molecular mechanism of EN against cancer has still not been elucidated. In this study, we examined the regulatory effect of EN on autophagy to reveal the mechanism of EN-mediated colon cancer growth inhibition. First, we found that EN is able to inhibit colon cancer cell proliferation and colony formation. The angiogenesis level in cancer cells was inhibited as well. Next, the treatment of EN led to the suppression of autophagy, which was characterized by the downregulation of the LC3-II level and the formation of GFP-LC3 puncta under EN treatment in colon cancer. Moreover, we revealed that the mTOR signaling pathway was activated by EN in a time- and concentration-dependent manner. Finally, autophagy induction protected colon cancer cells from EN treatment, suggesting that autophagy improves cell survival. Taken together, our findings revealed the mechanism of EN against colon cancer through inhibiting autophagy and angiogenesis in colon cancer, supporting that the autophagy inhibitor EN could be developed to be a novel anti-cancer agent.     

Co-Chaperone Bag-1 Plays a Role in the Autophagy-Dependent Cell Survival through Beclin 1 Interaction

Expression levels of the major mammalian autophagy regulator Beclin 1 and its interaction with Bcl-2 regulate the switch between autophagic cell survival and apoptotic cell death pathways. However, some of the regulators and the precise mechanisms of these processes still remain elusive. Bag-1 (Bcl-2 associated athanogene-1), a member of BAG family proteins, is a multifunctional pro-survival molecule that possesses critical functions in vital cellular pathways. Herein, we report the role of Bag-1 on Bcl-2/Beclin 1 crosstalk through indirectly interacting with Beclin 1. Pull-down experiments suggested a molecular interaction between Bag-1 and Beclin 1 in breast cancer cell lines. On the other hand, in vitro binding assays showed that Bag-1/Beclin 1 interaction does not occur directly but occurs through a mediator molecule. Bag-1 interaction with p-Beclin 1 (T119), indicator of early autophagy, is increased during nutrient starvation suggesting involvement of Bag-1 in the autophagic regulation. Furthermore, CRISPR/Cas9-mediated Bag-1 knock-out in MCF-7 cells hampered cell survival and proliferation and resulted in decreased levels of total LC3 under starvation. Collectively, we suggest that Bag-1 modulates cell survival/death decision through maintaining macroautophagy as a component of Beclin 1-associated complexes.     

The death of human cancer cells following photodynamic therapy: apoptosis competence is necessary for Bcl-2 protection but not for induction of autophagy

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.     

Davidone C Induces the Death of Hepatocellular Carcinoma Cells by Promoting Apoptosis and Autophagy

Davidone C is a newly discovered flavonoid compound purified from the ethyl acetate-soluble fraction of Sophora davidii (Franch.) Skeels. This study explored the anti-tumor activity of davidone C on hepatocellular carcinoma HepG2 and Bel-7402 cells and its mechanism through MTT method, morphological observation, flow cytometry and Western blotting. The results showed that davidone C significantly inhibited the proliferation of HepG2 and Bel-7402 cells in a time- and dose-dependent manner. The morphological changes of apoptotic cells can be observed under an inverted microscope, such as cell floating, chromosome condensation, apoptotic bodies, and other phenomena. The expressions of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP increased with the increase of dosage while Bcl-2 decreased, suggesting that the apoptotic mechanism might be related to the mitochondrial apoptotic pathway. Moreover, davidone C administration can down-regulate the expression of Grp78, and simultaneously up-regulate the expression of caspase-7 and caspase-12, indicating that the apoptotic mechanism might be related to the ERS pathway. In addition, davidone C can down-regulate the expression of p62, and simultaneously up-regulate the expression of LC3-I and LC3-II with a quantitative dependence, suggesting that the mechanism of apoptosis may be related to the autophagy signal pathway. All these results showed davidone C has potential effects on hepatocellular carcinoma.     

Lycium barbarum polysaccharide with potential anti-gastric cancer effects mediated by regulation of miR-202-5p/PIK3CA

Lycium barbarum polysaccharide (LBP) in addition to modifying inorganic nanoparticles shows different biological functions such as anti-cancer, antibacterial, and anti-aging performances. However, the potential mechanism of LBP on inhibition of cancer cell proliferation, particularly gastric cancer (GC), remains unknown. The goal of this study was to show how LBP induces its anti-cancer effects through regulation of the miR-202-5p/PIK3CA axis in GC. The MTT assay was used to assess the viability of AGT and GES-1 cells. Using quantitative real-time PCR we assessed miR-202-5p expression in AGS, BCG-823, GES-1, MKN-45, and SGC-790a cells. AGS cells were transfected with miR-202-5p, an inhibitor, and a small interfering RNA (siRNA) targeting PIK3CA. To show whether miR-202-5p directly targets PIK3CA, the luciferase reporter assay was used. Also, to assess protein levels of PIK3CA/AKT/mTOR, Bax/Bcl-2, Cleaved Caspase-3, and MMP9 and GC cell migration ability, western blot and transwell assays were used, respectively. The results showed that LBP decreased GC cell viability in a dose- and time-dependent manner. Furthermore, GC cell treatment with LBP substantially decreased cell proliferation and migration, while increased GC cell apoptosis. LBP induced the upregulation of caspase-3/7 and miR-202-5p in GC cells and directly and functionally targets PIK3CA, as verified by luciferase assay and anti-miR-202-5p’s capability to reverse the inhibitory effects of LBP on PIK3CA. LBP was also shown to decrease the expression of PIK3CA downstream members such as AKT and mTOR through miR-202-5p up-regulation. Anti-cancer properties of LBP in GC cells are possibly due to the up-regulation of miR-202, which inhibits the PIK3CA/AKT/mTOR axis.     

Wheat Germ Spermidine and Clove Eugenol in Combination Stimulate Autophagy In Vitro Showing Potential in Supporting the Immune System against Viral Infections

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.     

Cure of human diabetic neuropathy by HPLC validated bark extract of Onosma echioides L. root

HPLC validated hexane bark extract of Onosma echioides L. root (OE) was evaluated for cure of human diabetic neuropathy in human neuroblastoma cell line. HPLC analysis was performed. Human neuroblastoma cells were grouped into control, normal glucose, high glucose (HG) and HG plus different concentrations of OE extract (10, 25 and 50 μg/mL). MTT, DCFH-DA staining and nuclear condensation assays were performed on neuroblastoma cells to evaluate antiproliferative activity, ROS activity level and apoptotic effect of OE. HPLC analysis revealed the existence of maximum yield of shikonin in n-hexane extract of OE. Exposure with different concentrations of OE effectively decreased ROS level and apoptosis of cells and as a result improved the viability of cells in a dose dependent manner in response to HG-induced oxidative stress. Thus, OE possesses the property to cure human diabetic neuropathy and further can be clinically tested for its use in diabetic neuropathy.     

The Ameliorative Effect of Empagliflozin in Vigabatrin-Induced Cerebellar/Neurobehavioral Deficits: Targeting mTOR/AMPK/SIRT-1 Signaling Pathways

Introduction. Vigabatrin (VGB) is an antiepileptic drug that acts to irreversibly inhibit the γ-aminobutyric acid (GABA) transaminase enzyme, elevating GABA levels. Broad studies have established that long-term treatment and/or high doses of VGB lead to variable visual defects. However, little attention has been paid to its other side effects, especially those demonstrating cerebellar involvement. Sodium glucose-linked co-transporter 2 (SGLT2) inhibitors are antidiabetic agents with protective effects far greater than expected based on their anti-hyperglycemic effect. Method. Our study herein was designed to investigate the possible ameliorative effect of empagliflozin, the SGLT2 inhibitors, in VGB-induced cerebellar toxicity. A total of 40 male Wistar rats were allocated equally into 4 groups: Group I: control group; Group II: VGB group; Group III empagliflozin treated VGB group; and Group IV: empagliflozin treated group. All groups were subjected to the detection of cerebellar messenger RNA gene expression of silent mating type information regulation 2 homolog 1 (SIRT1) and Nucleoporin p62 (P62). Mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase (AMPK), and beclin1 levels were assessed by the ELISA technique while malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected spectrophotometrically. Immuno-histochemical studies, focusing on glial fibrillary acidic protein (GFAP) and S100 were performed, and the optical color density and the mean area percentage of GFAP positive astrocytes and the number of S 100 positive cells were also counted. Results. Following empagliflozin treatment, we documented significant upregulation of both SIRT1 and P62 mRNA gene expression. Additionally, AMPK, Beclin1 levels, and SOD activity were significantly improved, while both mTOR and MDA levels were significantly reduced. Conclusions. We concluded for the first time that empagliflozin efficiently ameliorated the VGB-induced disrupted mTOR/AMPK/SIRT-1 signaling axis with subsequent improvement of the autophagy machinery and mitigation of the oxidative and inflammatory cellular environment, paving the way for an innovative therapeutic potential in managing VGB-induced neurotoxicity.     

Nigrosporins B,a Potential Anti-Cervical Cancer Agent,Induces Apoptosis and Protective Autophagy in Human Cervical Cancer Ca Ski Cells Mediated by PI3K/AKT/mTOR Signaling Pathway

Nigrosporins B, an anthraquinone derivative obtained from the secondary metabolites of marine fungus Nigrospora oryzae. In this study, we characterized the distinctive anti-cancer potential of Nigrosporins B in vitro and underlying molecular mechanisms in human cervical cancer Ca Ski cells for the first time. The results of MTT assay showed that Nigrosporins B significantly inhibited the proliferation of multiple tumor cells in a dose-dependent manner, especially for the Ca Ski cells with an IC₅₀ of 1.24 µM. Nigrosporins B exerted an apoptosis induction effect on Ca Ski cells as confirmed by flow cytometry, AO/EB dual fluorescence staining, mitochondrial membrane potential analysis and western blot assay. In addition, Nigrosporins B induced obvious autophagy accompanied with the increase of autophagic vacuoles and the acceleration of autophagic flux as indicated by Cyto-ID staining, mRFP-GFP-LC3 adenovirus transfection and western blot analysis. Interestingly, the combination of Nigrosporins B with the three autophagy inhibitors all significantly enhanced the cytotoxicity of Nigrosporins B on Ca Ski cells, indicating that the autophagy induced by Nigrosporins B might protect Ca Ski cells from death. Furthermore, we found that Nigrosporins B inhibited the phosphorylation of PI3K, AKT, mTOR molecules and increased the protein expression levels of PTEN and p-AMPKα in a dose-dependent manner, suggesting that Nigrosporins B induced apoptosis and protective autophagy through the suppression of the PI3K/AKT/mTOR signaling pathway. Together, these findings revealed the anti-cervical cancer effect of Nigrosporins B and the underlying mechanism of action in Ca Ski cells, it might be as a promising alternative therapeutic agent for human cervical cancer.     

Andrographolide Exhibits Anticancer Activity against Breast Cancer Cells (MCF-7 and MDA-MB-231 Cells) through Suppressing Cell Proliferation and Inducing Cell Apoptosis via Inactivation of ER-α Receptor and PI3K/AKT/mTOR Signaling

Breast cancer is the most common cancer among women worldwide. Chemotherapy followed by endocrine therapy is the standard treatment strategy after surgery or radiotherapy. However, breast cancer is highly resistant to the treatments leading to the recurrence of breast cancer. As a result, the development of alternative medicines derived from natural plants with fewer side effects is being emphasized. Andrographolide isolated from Andrographis paniculata is one of the potential substances with anti-cancer properties in a variety of cell types, including breast cancer cells. This study aims to investigate the anti-cancer effects of andrographolide in breast cancer cells by evaluating cell viability and apoptosis as well as its underlying mechanisms through estrogen receptor (ER)-dependent and PI3K/AKT/mTOR signaling pathways. Cell viability, cell apoptosis, mRNA or miRNA, and protein expression were examined by MTT assay, Annexin V-FITC, qRT-PCR, and Western blot analysis, respectively. MCF-7 and MDA-MB-231 cell viability was reduced in a concentration- and time-dependent manner after andrographolide treatment. Moreover, andrographolide induced cell apoptosis in both MCF-7 and MDA-MB-231 cells by inhibiting Bcl-2 and enhancing Bax expression at both mRNA and protein levels. In MCF-7 cells, the ER-positive breast cancer, andrographolide showed an inhibitory effect on cell proliferation through downregulation of ERα, PI3K, and mTOR expression levels. Andrographolide also inhibited MDA-MB-231 breast cancer cell proliferation via induction of cell apoptosis. However, the inhibition of MCF-7 and MDA-MB-231 cell proliferation of andrographolide treatment did not disrupt miR-21. Our findings showed that andrographolide possesses an anti-estrogenic effect by suppressing cell proliferation in MCF-7 cells. The effects were comparable to those of the anticancer drug fulvestrant in MCF-7 cells. This study provides new insights into the anti-cancer effect of andrographolide on breast cancer and suggests andrographolide as a potential alternative from the natural plant for treating breast cancer types that are resistant to tamoxifen and fulvestrant.     

In Vivo Effect of Resveratrol-Loaded Solid Lipid Nanoparticles to Relieve Physical Fatigue for Sports Nutrition Supplements

Resveratrol (RSV) is a natural flavonoid polyphenol compound extracted from the plants which shows various biological activities. However, the clinical application of RSV is limited by its poor aqueous solubility, rapid metabolism and poor bioavailability. In this study, resveratrol-loaded solid lipid nanoparticles (RSV- SLNs) was design as a nano-antioxidant against the physical fatigue. The resultant RSV-SLNs were characterized by photon correlation spectroscopy (PCS), transmission electron micrographs (TEM), zeta potential, differential scanning calorimetry (DSC) and Raman spectroscopy pattern. Furthermore, the in vivo anti-fatigue effect assays showed that RSV-SLNs prolonged the mice exhausted time and running distance. The biochemical parameters of blood related to fatigue suggested that RSV-SLNs have potential applications to improve the antioxidant defense of the mice after extensive exercise and confer anti-fatigue capability. Furthermore, the molecular mechanisms of antioxidant by RSV-SLNs supplementation was investigated through the analysis of silent information regulator 2 homolog 1 (SIRT1) protein expression, which demonstrated that it could downregulate the expression of SIRT1 and increase autophagy markers, microtubule-associated protein 1 light chain 3-II (LC3-II) and sequestosome-1 (SQSTM1/p62). These results reveal that the RSV-SLNs may have great potential used as a novel anti-fatigue sports nutritional supplement.     

The role of autophagy in cadmium-induced acute toxicity in glomerular mesangial cells and tracking polyubiquitination of cytoplasmic p53 as a biomarker

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development.Subject terms: Macroautophagy, Diagnostic markers