scholarly journals 764 Characterization of RVU-27065 a novel small-molecule STING agonist suitable for systemic administration

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A799-A799
Author(s):  
Maciej Rogacki ◽  
Stefan Chmielewski ◽  
Magdalena Zawadzka ◽  
Aniela Gołas ◽  
Aleksandra Poczkaj ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Growth ◽  
Western Blot ◽  
Cell Viability ◽  
Small Molecule ◽  
Blot Analysis ◽  
Targeted Delivery ◽  
Western Blot Analysis ◽  
Dual Reporter

BackgroundSTimulator of INterferon Genes (STING) is a key signaling protein involved in activation of the immune system in response to self-DNA. In recent years, STING signaling has been demonstrated to play a major role in activating the antitumor immune response and therefore is considered an attractive drug target in immuno-oncology. The first wave of STING agonists, cyclic-dinucleotide analogues of the internal ligand cGAMP, were developed for local, intratumoral administration. Herein we present the most recent profiling results of our frontrunner molecule RVU-27065, a potent and selective systemic STING agonist with a favorable drug profile.MethodsBinding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation. Primary activity screen was performed in THP-1 Dual reporter cells. Selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing STING variants. T cell viability and proliferation was assessed by flow cytometry using activated human T cells. PBMCs were isolated by density gradient from whole blood of healthy donors. Downstream STING pathway activation in cells treated with RVU-27065 was confirmed using Western blot analysis. BALB/c mice were inoculated with EMT6 tumor cells and the compound was administered intravenously followed by regular monitoring of tumor growth. Cured animals were rechallenged by repeated inoculation of EMT6 cells.ResultsRVU-27065 binds and strongly thermostabilizes recombinant STING proteins of all tested species. Binding to the protein results in activation of downstream signalling pathway, confirmed by western blot analysis. The agonist is characterized by selectivity and excellent potency in THP-1 dual reporter cells as well as in human PBMCs and dendritic cells. Short term incubation of RVU-27065 has no impact on T cell viability, activation or proliferation. Furthermore, STING activation with RVU-27065 leads to repolarization of immunosuppressive M2 macrophages into pro-inflammatory M1-like phenotype. In vivo efficacy of RVU-27065 was confirmed, leading to significant tumor growth inhibition and complete tumor regressions in an EMT6 mouse breast cancer syngeneic tumor model.ConclusionsRVU-27065 is a novel representative of a 3rd generation of Ryvu STING agonists – small-molecule, non-macrocyclic molecules built around a unique chemotype. The compound is characterized by high in vitro potency which translates to efficacy in vivo in preclinical animal models. Drug-like properties, excellent selectivity and a good safety profile make RVU-27065 an attractive candidate for further development for standalone as well as targeted delivery, which holds high potential for improved immunotherapy in cancer patients.

Effect of trametinib in combination with panitumumab and trastuzumab on tumor growth in an orthotopic xenograft model of human pancreatic cancer.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 190-190
Author(s):  
James M. Lindberg ◽  
Sara J Adair ◽  
Timothy E. Newhook ◽  
Alison Kim ◽  
J Thomas Parsons ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Western Blot ◽  
Growth Inhibition ◽  
Triple Therapy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ras Pathway

190 Background: Aberrant MAPK and EGFR family signaling are key drivers of pancreatic ductal adenocarcinoma(PDAC). We hypothesized that combination trametinib(MEK1/2 inhibitor), panitumumab(EGFR inhibitor) and trastuzumab(Her2 inhibitor) would more effectively suppress tumor growth than any of these monotherapies. Methods: Patient-derived PDAC cell line MAD09-366 was exposed to trametinib, panitumumab, trastuzumab, and combination therapies in vitro. Western blot analysis was performed on treated cell lysates. Athymic, nude mice were orthotopically implanted with patient-derived PDAC xenografts(MAD09-366, 08-608, and 08-738). Established murine tumors were treated with control, trametinib (0.3mg/kg, qDay), panitumumab (500ug, BIW), trastuzumab (200ug, BIW) or in combination. MRI was used to assess tumor response. Results: Two of 3 PDACs were Kras mutant, 2 of 3 demonstrated increased Her2 activity, and all 3 showed increased EGFR activity. In vitro studies showed increased growth inhibition of triple-therapy-treated cells relative to control or each inhibitor alone. Western blot analysis revealed that EGF stimulation increased Ras pathway signaling in this Kras-mutant cell line. With EGF stimulation, the greatest Ras pathway signaling inhibition was seen in triple-therapy-treated cells. In vivo studies in all PDAC xenografts revealed that triple therapy significantly decreased tumor growth rate relative to control, trametinib alone, panitumumab alone, or panitumumab plus trastuzumab. In 2 of 3 PDACs assessed, triple therapy was superior to trametinib plus panitumumab. Average tumor size in MAD08-738 triple-therapy-treated mice decreased by 9.3%. Conclusions: Triple therapy with trametinib, panitumumab, and trastuzumab demonstrated the greatest in vitro Ras signaling blockade. In vivo, this combination produced significant tumor growth inhibition or regression in all PDAC tumors studied. This regimen should be considered for a future clinical trial in pancreatic cancer patients.

scholarly journals Paeonol Induces Protective Autophagy in Retinal Photoreceptor Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Daowei Zhang ◽  
Jiawen Wu ◽  
Jihong Wu ◽  
Shenghai Zhang
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Retinal Photoreceptor

Background: Retinal photoreceptor (RP) cells are widely involved in retina-related diseases, and oxidative stress plays a critical role in retinal secondary damage. Herein, we investigated the effectiveness and potential mechanisms of autophagy of paeonol (Pae) in terms of oxidation resistance.Methods: The animal model was induced by light damage (LD) in vivo, whereas the in vitro model was established by H2O2 stimulation. The effectiveness of Pae was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluorescence, transmission electron microscopy, electroretinogram, and Western blot analysis in vivo, and the underlying mechanisms of Pae were assessed by Cell Counting Kit-8 assay, reactive oxygen species (ROS) assay, and Western blot analysis in 661W cells. We mainly evaluated the effects of Pae on apoptosis and autophagy.Results: Increased apoptosis of the LD-induced and decreased autophagy of RPs were mitigated by Pae treatment. Pea, which increased the expression of mitochondrial functional protein cytochrome c, reversed the decreased cell viability and autophagy induced by oxidative stress in 661W cells. Experiments showed that autophagy was downregulated in PINK1/Parkin dependent and the BNIP3L/Nix dependent pathways under H2O2 stimulation and was upregulated by Pae treatment. Pae increased the cell viability and reduced ROS levels through autophagy.Conclusion: Pretreatment with Pae preserved RP cells by enhancing autophagy, which protected retinal function.

scholarly journals RUNX2 Plays An Oncogenic Role in Esophageal Carcinoma by Activating the PI3K/AKT and ERK Signaling Pathways

2018 ◽  
Vol 49 (1) ◽  
pp. 217-225 ◽  
Author(s):  
Huibin Lu ◽  
Tian Jiang ◽  
Kewei Ren ◽  
Zongming Li Li ◽  
Jianzhuang Ren ◽  
...  
Keyword(s):  
Western Blot ◽  
Esophageal Carcinoma ◽  
Cell Viability ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tumor Formation ◽  
Erk Signaling ◽  
Migration And Invasion

Background/Aims: Esophageal carcinoma is a frequently occurring cancer at upper gastrointestinal tract. We aimed to evaluate the roles and possible mechanism of Runt Related Transcription Factor 2 (RUNX2) in the development of esophageal cancer. Methods: The expression of RUNX2 in esophageal carcinoma tissues and cells was investigated by qRT-PCR. Effects of RUNX2 on cell viability, apoptosis, migration and invasion were assessed using MTT assay, flow cytometry assay/western blot analysis, and Transwell assays, respectively. Afterwards, effects of RUNX2 on of the activation of the PI3K/AKT and ERK pathways were explored by Western blot analysis. In addition, a PI3K/AKT pathway inhibitor LY294002 and an ERK inhibitor U0126 were applied to further verify the regulatory relationship between RUNX2 and the PI3K/AKT and ERK signaling pathways. Besides, the RUNX2 function on tumor formation in vivo was investigated by tumor xenograft experiment. Results: The result showed that RUNX2 was highly expressed in esophageal carcinoma tissues and cells. Knockdown of RUNX2 significantly inhibited TE-1 and EC-109 cell viability, repressed TE-1 cell migration and invasion, and increased TE-1 cell apoptosis. RUNX2 overexpression showed the opposite effects on HET-1A cells. Moreover, RUNX2-mediated TE-1 cell viability, migration and invasion were associated with the activation of the PI3K/AKT and ERK pathways. Besides, knockdown of RUNX2 markedly suppressed tumor formation in vivo. Conclusion: Our results indicate that RUNX2 may play an oncogenic role in esophageal carcinoma by activating the PI3K/ AKT and ERK pathways. RUNX2 may serve as a potent target for the treatment of esophageal carcinoma.

scholarly journals Therapeutic Potential of Axl Blockade in BCR-ABL Negative Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3063-3063
Author(s):  
Antonia Beitzen-Heineke ◽  
Isabel Ben Batalla ◽  
Nikolaus Berenbrok ◽  
Sarina Paesler ◽  
Victoria Gensch ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Therapeutic Potential ◽  
Brdu Incorporation ◽  
Additive Effects ◽  
Healthy Donors

Abstract Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases. We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p<0.001) and BaF3-Jak2V617F cells (increase by 54% at 2µM, p<0.05). Moreover, Western Blot analysis showed higher levels of cleaved caspase 3 in BGB324 treated SET-2 cells and decreased levels of anti-apoptotic bcl-2 in BGB324 treated BaF3-Jak2V617F cells. Additionally, BrdU incorporation assays showed a dose dependent decrease in proliferating cells upon treatment with BGB324 in MPN cell lines (p<0.05). Genetic knockdown of Axl in SET-2 cells decreased cell viability by 75% (p<0.01), increased apoptosis levels as measured by Annexin+ staining by 61% (p<0.05) and decreased proliferation as measured by BrdU incorporation by 35% (p<0.001) compared to control-transduced cells. Furthermore, Western Blot analysis revealed that genetic knockdown of Axl resulted in decreased phosphorylation of Stat3 and Stat5 compared to control-transduced cells. Combined Axl and Jak2 blockade, using BGB324 and the Jak2-inhibitor ruxolitinib, showed additive effects on reducing cell viability in SET-2 and BaF3-Jak2V617F cells (p<0.01 and p<0.001, respectively). Western Blot analysis identified inhibition of Stat5 by BGB324 single treatment in SET-2 cells whereas additive effects of combined Axl and Jak2 blockade resulted from additional inhibition of Stat3. In BaF3-Jak2V617F cells, BGB324 single treatment resulted in downstream inhibition of Akt signaling whereas additive effects of combined Axl and Jak2 blockade were exerted via additional inhibition of Stat5, Stat3 and Erk. The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p<0.01), with a 60% reduction of tumor weight compared to vehicle treated mice (n=8/8, p<0.01). As a second in vivo model, a systemic model of Jak2V617F driven disease was used. After intravenous injection of BaF3-Jak2V617F cells, mice were treated with 50mg/kg BGB324 or vehicle starting the day after inoculation. BGB324 treated mice had a longer overall survival compared to vehicle treated mice (n=10/11, p*<0.05). Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p<0.001) or 2µM (100% vs. 60%, respectively; p<0.01) of BGB324 (n=5/5). In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation. Disclosures von Amsberg: Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals MicroRNA-145 attenuates IL-6-induced enhancements of sensitivity to UVB irradiation by suppressing MyD88 in HaCaT cells

2018 ◽  
Vol 32 ◽  
pp. 205873841879594 ◽  
Author(s):  
Hui Dong ◽  
Wei Jiang ◽  
Hongquan Chen ◽  
Shui Jiang ◽  
Yunshu Zang ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Lupus Erythematosus ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Ultraviolet B ◽  
Hacat Cells ◽  
Related Factors ◽  
Uvb Irradiation

MicroRNAs (miRNAs/miRs) play vital roles in various immune diseases including systemic lupus erythematosus (SLE). The current study aimed to assess the role of miR-145 in interleukin-6 (IL-6)-treated HaCaT cells under ultraviolet B (UVB) irradiation and further explore the potential regulatory mechanism. HaCaT cells were pretreated with IL-6 and then exposed to UVB to assess the effect of IL-6 on sensitivity of HaCaT cells to UVB irradiation. The levels of miR-145 and MyD88 were altered by transfection and the transfected efficiency was verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR)/western blot analysis. Cell viability, percentage of apoptotic cells and expression levels of apoptosis-related factors were measured by trypan blue assay, flow cytometry assay, and western blot analysis, respectively. In addition, the levels of c-Jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) signaling pathway-related factors were assessed by western blot analysis. IL-6 treatments significantly aggravated the reduction of cell viability and promotion of cell apoptosis caused by UVB irradiation in HaCaT cells. Interestingly, miR-145 level was augmented by UVB exposure and miR-145 mimic alleviated IL-6-induced increase of sensitivity to UVB irradiation in HaCaT cells, as dramatically increased cell viability and reduced cell apoptosis. Opposite effects were observed in miR-145 inhibitor-transfected cells. Meanwhile, MyD88 was negatively regulated by miR-145 and MyD88 mediated the regulatory effect of miR-145 on IL-6- and UVB-treated cells. In addition, miR-145 mimic inhibited the JNK and NF-κB pathways by down-regulating MyD88. In conclusion, the present study demonstrated that miR-145 alleviated IL-6-induced increase of sensitivity to UVB irradiation by down-regulating MyD88 in HaCaT cells.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

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