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

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

Journal of Neurosurgery ◽

10.3171/2010.7.gks10972 ◽

2010 ◽

Vol 113 (Special_Supplement) ◽

pp. 228-235 ◽

Cited By ~ 4

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.

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

10.21203/rs.3.rs-88666/v1 ◽

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.

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

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17238811 ◽

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.

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ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Blood ◽

10.1182/blood.v104.11.126.126 ◽

2004 ◽

Vol 104 (11) ◽

pp. 126-126 ◽

Cited By ~ 2

Author(s):

Weirui Zhang ◽

David Motto ◽

David Ginsburg

Keyword(s):

Fusion Protein ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Expression Vector ◽

Full Length ◽

Mammalian Expression ◽

Partial Denaturation

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

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

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.190 ◽

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.

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Potent In Vivo Anti-Tumor Activity Of Extracellular Vesicles Isolated From Genetically Engineered Primary Mesenchymal Stromal Cells Expressing The Trans-Membrane TNF-Related Apoptosis-Inducing Ligand (TRAIL)

Blood ◽

10.1182/blood.v122.21.1658.1658 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1658-1658

Author(s):

Stefano Buttiglieri ◽

Carmelo Carlo-Stella ◽

Tiziana Spatola ◽

Roberta Pulito ◽

Luigi Naldini ◽

...

Keyword(s):

Western Blot ◽

Tumor Cells ◽

Stromal Cells ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cytotoxic Effect ◽

Genetically Engineered ◽

Nsg Mice

Abstract Introduction TNF-related apoptosis-inducing ligand (TRAIL) is a protein functioning as a ligand that induces the process of cell death. TRAIL has been shown to kill in vitro a wide variety of tumor cells with minimal effects on normal cells. Despite its in vitro activity, recombinant soluble TRAIL has so far shown limited efficacy in vivo. In contrast, recent reports have shown that significant apoptosis can be observed both in vitro and in vivo when TRAIL is expressed on the cell membrane (mTRAIL). A further innovation might be the delivery of bioactive proapoptotic TRAIL through its expression by extracellular vescicles (EVs), the nanovesicular organelles secreted by cells. In fact, EVs are viewed as an effective tool for intercellular cross-talk and receptor discharge. The trans-membrane expression of TRAIL ligand within the double layer exosomal membrane may induce a more potent death signal when compared with the soluble molecule. Material and Methods Mesenchymal Stromal Cells (MSC) from bone marrow were cultured in vitro and used for EVs production. Cultured MSC in 75 cm2 flasks, at 80% confluence were infected with a lentivector encoding TRAIL, maintained in culture, and cell-supernatants repeatedly collected over several days, ultracentrifugated, with EVs-containing pellet harvested in PBS. EVs were produced also from uninfected MSC as control (EVs-CTRL). EVs were characterized by flow cytometry for expression of MSC markers and mTRAIL, EV size was evaluated by NanoSight technology. Total protein concentration was used to quantify EVs, Western Blot analysis was performed to characterize membrane-bound TRAIL. In vitro analysis was performed on SU-DHL-4 (human B cell lymphoma) and MEL-1300 (human melanoma) cell lines, exposed for 24 hours to 20-100 μg/ml EVs-TRAIL or EVs-CTRL. Annexin/propidium iodide assay was used to quantify apoptotic/necrotic cells. For the in vivo assessments, SU-DHL-4 and MEL-1300 cells were transduced with Luc-Lentiviral particles to obtain Luciferase positive cell lines. These cells were used to engraft NOD scid gamma (NSG) mice (2x106 SU-DHL-4 and 3x105 MEL-1300 cells for each subcutaneous injection point). To visualize tumor cells, mice were injected intraperitoneum with luciferin and analyzed with the Xenogen system. Mice bearing subcutaneous tumor nodules received single intravenous injections of 100, 200, 300 µg or multiple (x 3) 200 µg injections of either EVs-TRAIL or EVs-CTRL. Results FACS analysis showed strong TRAIL expression on EVs from TRAIL-infected MSC compared to EVs-CTRL, with a high proportion of positive particles (median 85%, range 78-93). In addition, EVs-TRAIL displayed MSC membrane markers, i.e. CD 105, CD 90, CD73 and CXCR4. Western Blot analysis under non-reducing conditions showed the presence of TRAIL ligand, with strong prevalence of dimeric TRAIL isoform (barely detectable the trimeric isoform, undetectable monomeric isoforms). NanoSight analysis revealed that EVs had a variable size, up to approximately 400 nm in diameter, with a predominant peak at 273 nm. A strong and dose-dependent cytotoxic effect was observed on SU-DHL-4 cells exposed to EVs-TRAIL (annexin/PI+ve cells: up to 87% for 100 μg/ml EVs-TRAIL), compared to EVs-CTRL exposure (15% Annexin/PI+ve cells for 100 μg/ml EVs-TRAIL). A similar, albeit less pronounced in vitro cytotoxic effect of EVs-TRAIL was observed on the melanoma MEL-1300 cell line. The anti-tumor effect was remarkably strong when EVs-TRAIL were injected in vivo in mice bearing either SU-DHL-4 or MEL-1300 nodules. A marked reduction of the tumor luminescence from 1.2x1010 photon/sec to <108 photon/sec was observed at seven days since a single EVs-TRAIL injection at 200 and 300 μg. Multiple administrations of 200 μg EVs-TRAIL induced the strongest luminescence reduction, as observed in MEL-1300 bearing NSG mice. Histological examination of nodules from EVs treated mice showed necrosis areas along with extensive intra-tumor vascular disruption. Conclusion EVs isolated from genetically engineered TRAIL-expressing MSC: i. do express mTRAIL; ii. display potent antitumor activity, inducing extensive apoptosis/necrosis both in vitro and in vivo in animal models bearing lymphoma and melanoma nodules. Thus, EVs-TRAIL may represent a promising strategy for delivering pro-apoptotic signals to tumor cells. Moreover, the Results could pave the way to the use of EVs for therapeutic purposes. Disclosures: No relevant conflicts of interest to declare.

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Does Valproic Acid Induce Neuroendocrine Differentiation in Prostate Cancer?

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/607480 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Abhinav Sidana ◽

Muwen Wang ◽

Wasim H. Chowdhury ◽

Antoun Toubaji ◽

Shabana Shabbeer ◽

...

Keyword(s):

Prostate Cancer ◽

Valproic Acid ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Neuroendocrine Differentiation ◽

Immunohistochemical Analysis ◽

Dependent Manner

Valproic Acid (VPA) is a histone deacetylase inhibitor that holds promise for cancer therapy. Here, we investigate whether VPA treatment induces neuroendocrine differentiation of Prostate Cancer (PCa). A tissue microarray of VPA-treated and untreated tumor xenografts and cell lines of human PCa (LNCaP, C4-2, DU145, and PC-3) were generated and were analyzed by immunohistochemical analysis (IHC) for NE markers chromogranin A (CgA), synaptophysin, and NCAM (neural cell adhesion molecule). Western blot analysis for CgA was performed to confirm the results of the TMA. IHC analysis did not reveal any induction of CgA, synaptophysin, or NCAM in any xenograft after VPA treatmentin vivo.In vitro, VPA treatment induced little synaptophysin expression in C4-2 and PC-3 cells and NCAM expression in LNCaP and PC-3 cells. In the case of CgA, VPA treatment decreased its expressionin vitroin a dose-dependent manner, as determined by western blot analysis. Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevantin vivosetting.

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In Vivo Evaluation of Novel Geranylgeranyl Diphosphate Synthase Inhibitors

Blood ◽

10.1182/blood-2018-99-113007 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 215-215 ◽

Cited By ~ 1

Author(s):

Staci L. Haney ◽

Yashpal S. Chhonker ◽

Michelle L. Varney ◽

Geoffrey A. Talmon ◽

Daryl J. Murry ◽

...

Keyword(s):

Single Dose ◽

Western Blot ◽

Blot Analysis ◽

Half Life ◽

Western Blot Analysis ◽

Weekly Schedule ◽

Geranylgeranyl Diphosphate Synthase ◽

Geranylgeranyl Diphosphate

Abstract The enzyme geranylgeranyl diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid geranylgeranyl diphosphate which is used in protein geranylgeranylation reactions. Our work has demonstrated that GGDPS inhibitors (GGDPSIs) represent a novel therapeutic strategy for multiple myeloma (MM) by disrupting Rab protein geranylgeranylation. Treatment of MM cells with GGDPSI results in disruption of monoclonal protein trafficking, leading to induction of the unfolded protein response pathway (UPR) and apoptosis. We have previously reported preclinical studies with a lead GGDPSI, VSW1198 (a mixture of homogeranyl/homoneryl triazole bisphosphonates), demonstrating the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 47.7 (±7.4) hrs), systemic distribution and confirmed in vivo disruption of geranylgeranylation (Haney et al., Invest New Drugs, 2018). Additional structure-function efforts have led to the development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). Intriguingly the addition of the α-methyl group abrogates the effects of the olefin stereochemistry on inhibitor potency such that the individual isomers display near identical ability to disrupt protein geranylgeranylation in enzyme and cell assays (Matthiesen et al., Bioorg Med Chem, 2018). As little is known regarding the impact of olefin stereochemistry on the pharmacokinetic (PK)/pharmacodynamic (PD) properties of drugs, we pursued additional in vitro and in vivo studies of RAM2093 and RAM2061 and investigated the efficacy of the GGDPSIs in a mouse MM xenograft model. In MM cell lines, qRT-PCR and western blot analysis showed that both isomers induce activation of UPR/apoptotic markers in a dose-dependent manner and with similar potency. Single dose testing in CD-1 mice identified a maximum tolerated dose of 0.5 mg/kg IV for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation with both compounds. Analysis of blood samples showed elevated liver transaminase levels with normal bilirubin/alkaline phosphatase levels and histopathological examination confirmed evidence of hepatocyte injury at higher doses. Multi-dose schedules of 0.1 mg/kg twice a week (two weeks on, one week off, two weeks on) for RAM2061 and RAM2093, as well as 0.2 mg/kg (RAM2061) and 0.15 mg/kg (RAM2093) weekly x four weeks were tested. All dosing schedules were tolerated with the exception of the twice-weekly schedule for RAM2093. Consistent with the findings from our single dose testing, both multi-dose schedules induced transient elevation of hepatic transaminases. No loss of weight was observed and creatinine and CBC results were within normal limits for both single and multi-dose injected animals. Importantly, western blot analysis of mouse tissues collected from both RAM2061 and RAM2093 multi-dose-treated mice showed accumulation of unmodified Rap1a in the liver, kidney and spleen, indicating in vivo disruption of protein geranylgeranylation was achieved. Furthermore, using MM.1S MM cell flank xenographs in NOD-SCID mice, we observed a significant reduction in tumor growth in mice treated with either VSW1198 or RAM2061 relative to vehicle control. Lastly, PK/biodistribution studies with RAM2093 and RAM2061 were performed following a single dose of 0.3 mg/kg IV. Both compounds were detectable in plasma and liver samples for up to seven days post-injection demonstrating prolonged half-life and tissue distribution. The full PK parameters for both compounds will be presented and studies measuring drug levels in other tissues are ongoing. Taken together, these data suggest that RAM2061 and RAM2093 have equivalent anti-MM activity in vitro, but that RAM2061 is better tolerated in vivo. Whether this is a consequence of different PK properties vs. differences in tissue uptake or metabolism will be determined. These studies also confirm the in vivo efficacy of our novel GGDPSIs and support further development of these agents for the treatment of MM. Figure. Figure. Disclosures Holstein: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Localization, Regulation and Possible Consequences of Apoptotic Protease-Activating Factor-1 (Apaf-1) Expression in Granulosa Cells of the Mouse Ovary

Endocrinology ◽

10.1210/endo.140.6.6931 ◽

1999 ◽

Vol 140 (6) ◽

pp. 2641-2644 ◽

Cited By ~ 34

Author(s):

Rodolfo Robles ◽

Xiao-Jing Tao ◽

Alexander M. Trbovich ◽

Daniel V. Maravei ◽

Ravit Nahum ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Granulosa Cell ◽

Granulosa Cells ◽

Blot Analysis ◽

Cell Apoptosis ◽

Western Blot Analysis ◽

C Elegans

Abstract The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bc1-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.

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Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury

Scientific Reports ◽

10.1038/srep23472 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 9

Author(s):

Se-Yeon Lee ◽

Seahyoung Lee ◽

Eunhyun Choi ◽

Onju Ham ◽

Chang Youn Lee ◽

...

Keyword(s):

Cell Death ◽

Small Molecule ◽

Heart Function ◽

Ischemia Reperfusion ◽

Target Prediction ◽

Cardiac Cell ◽

Interacting Protein ◽

Genetic Ablation

Abstract Genetic ablation of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), an essential regulator of cardiac cell death, is an effective way to prevent cardiac cell death triggered by pathologic conditions. However, currently there exists no known means, such as inhibitors, to down-regulate BNIP3 in mature heart. Here, we report that a small molecule inducer of microRNA-182 (miR-182) suppressed ischemia/reperfusion (I/R)-induced cardiac cell death by down-regulating BNIP3. We first selected miR-182 as a potent BNIP3-targeting miRNA based on miRNA-target prediction databases and empirical data. The subsequent screening of small molecules for inducing miR-182 expression identified Kenpaullone as a hit compound. Both exogenous miR-182 and Kenpaullone significantly suppressed hypoxia-induced cardiomyocyte death in vitro. To investigate the effect of changing substituents of Kenpaullone on miR-182 expression, we synthesized 9 derivatives of Kenpaullone. Among these derivatives, compound 5 showed significantly improved ability to induce miR-182 expression. The results of the in vivo study showed that compound 5 significantly improved heart function following I/R-injury in rats. Our study provides strong evidence that the small molecule-mediated up-regulation of miRNAs is a viable strategy to down-regulate target proteins with no known chemical inhibitor and that compound 5 may have potential to prevent I/R-inflicted cardiac cell death.

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