scholarly journals Engineering of CHO cells for the production of vertebrate recombinant sialyltransferases

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e5788 ◽  
Author(s):  
Benoit Houeix ◽  
Michael T. Cairns
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Neuronal Development ◽  
Culture Media ◽  
Lectin Binding ◽  
Stable Cell Lines ◽  
Cell Matrix Interactions ◽  
Free Media

Background Sialyltransferases (SIATs) are a family of enzymes that transfer sialic acid (Sia) to glycan chains on glycoproteins, glycolipids, and oligosaccharides. They play key roles in determining cell–cell and cell-matrix interactions and are important in neuronal development, immune regulation, protein stability and clearance. Most fully characterized SIATs are of mammalian origin and these have been used for in vitro and in vivo modification of glycans. Additional versatility could be achieved by the use of animal SIATs from other species that live in much more variable environments. Our aim was to generate a panel of stable CHO cell lines expressing a range of vertebrate SIATs with different physicochemical and functional properties. Methods The soluble forms of various animal ST6Gal and ST3Gal enzymes were stably expressed from a Gateway-modified secretion vector in CHO cells. The secreted proteins were IMAC-purified from serum-free media. Functionality of the protein was initially assessed by lectin binding to the host CHO cells. Activity of purified proteins was determined by a number of approaches that included a phosphate-linked sialyltransferase assay, HILIC-HPLC identification of sialyllactose products and enzyme-linked lectin assay (ELLA). Results A range of sialyltransferase from mammals, birds and fish were stably expressed in CHO Flp-In cells. The stable cell lines expressing ST6Gal1 modify the glycans on the surface of the CHO cells as detected by fluorescently labelled lectin microscopy. The catalytic domains, as isolated by Ni Sepharose from culture media, have enzymatic activities comparable to commercial enzymes. Sialyllactoses were identified by HILIC-HPLC on incubation of the enzymes from lactose or whey permeate. The enzymes also increased SNA-I labelling of asialofetuin when incubated in a plate format. Conclusion Stable cell lines are available that may provide options for the in vivo sialylation of glycoproteins. Proteins are active and should display a variety of biological and physicochemical properties based on the animal source of the enzyme.

scholarly journals Effect of Jasmonic Acid and Salicylic Acid on Growth and Biochemical Composition of In-Vitro-Propagated Lavandula angustifolia Mill

Agronomy ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1722
Author(s):  
Ileana Miclea ◽  
Andreea Suhani ◽  
Marius Zahan ◽  
Andrea Bunea
Keyword(s):  
Salicylic Acid ◽  
Jasmonic Acid ◽  
Plant Material ◽  
Culture Media ◽  
Carotenoid Content ◽  
Lavandula Angustifolia ◽  
Beneficial Impact ◽  
Free Media

This study assessed the effect of jasmonic acid (JA) and salicylic acid (SA) on the in vitro development and production of Lavandula angustifolia Mill. plant material, and the accumulation of polyphenols, chlorophylls, and carotenoids in explants. Results were compared with explants grown in control media and with in-vivo-grown mature and young L. angustifolia plants. After 21 days of incubation, all explants propagated on low-SA-concentration or elicitor-free media produced a greater number of shoots than explants cultivated on media with higher elicitor concentrations. Shoots grew taller when activated charcoal (AC) was added to the elicitor-supplemented media, while AC negatively affected or had no effect on the phytochemical composition of plants. Explants grown in the presence of elicitors had higher polyphenolic and chlorophyll content than the controls, demonstrating the beneficial impact of elicitors on the secretion of secondary metabolites. Lutein and β-carotene were the dominating carotenoids in all samples. Culture media supplemented with 0.5 mg/L JA and 1.5 mg/L SA + AC proved the most suitable to produce plant material with high polyphenol and carotenoid content, comparable with in-vivo-grown plants.

scholarly journals Overexpression ofβ-Catenin Induces Cisplatin Resistance in Oral Squamous Cell Carcinoma

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Long Li ◽  
Hai-Chao Liu ◽  
Cheng Wang ◽  
Xiqiang Liu ◽  
Feng-Chun Hu ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
Cisplatin Resistance ◽  
Stable Cell Lines ◽  
Abnormal Expression

Abnormal expression ofβ-catenin contributes to tumor development, progression, and metastasis in various cancers. However, little is known about the relationship between abnormal expression ofβ-catenin and cisplatin chemotherapy in oral squamous cell carcinoma (OSCC). The present study aimed to investigate the effect ofβ-catenin on OSCC cisplatin resistance and evaluated the drug susceptibility of stable cell lines withβ-catenin knockin and knockdown. In this study, we found that higher expression level ofβ-catenin can be observed in CDDP-treated cell lines as compared with the control group. Furthermore, the expression levels ofβ-catenin increased in both a concentration- and time-dependent manner with the cisplatin treatment. More importantly, the nuclear translocation ofβ-catenin could also be observed by confocal microscope analysis. Stable cell lines with CTNNB1 knockin and knockdown were established to further investigate the potential role and mechanism ofβ-catenin in the chemoresistance of OSCC in vitro and in vivo. Our findings indicated that overexpression ofβ-catenin promoted cisplatin resistance in OSCC in vitro and in vivo. We confirmed that GSK-3β, C-myc, Bcl-2, P-gp, and MRP-1 were involved inβ-catenin-mediated drug resistance. Our findings indicate that the Wnt/β-catenin signaling pathway may play important roles in cisplatin resistance in OSCC.

CONTRIBUTION TO THE EXISTENCE OF REGULATORY MECHANISMS AT THE ADRENAL LEVEL

1972 ◽  
Vol 70 (4) ◽  
pp. 741-757
Author(s):  
Otto Linèt
Keyword(s):  
Orotic Acid ◽  
Adrenal Glands ◽  
Actinomycin D ◽  
Delay Period ◽  
Regulatory Mechanisms ◽  
Leucine Incorporation ◽  
Total Period ◽  
Free Media

ABSTRACT Rat adrenal glands atrophied by the administration of cortisol acetate in vivo were used as a model for the study of early metabolic processes occurring in vitro. Atrophied adrenals incubated in the presence of 14C-leucine incorporated subnormal quantities of this amino acid per mg of protein for the first 120 min. When the incubation lasted for a total period of 180 or 240 min a supranormal rise in the 14C-leucine incorporation was observed. Similar changes occurred with some delay with regard to corticosterone production as expressed per 100 mg of tissue. No differences in 14C-leucine incorporation were observed between the control and atrophied adrenals in vivo. Homogenates from atrophied glands incorporated 14C-leucine to a greater extent than the control homogenates. The in vitro incorporation of 14C-orotic acid into the RNA was also higher in atrophied adrenals. The in vitro use of actinomycin D, cycloheximide and amphenone indicated that corticosterone production depended on the incorporation of 14C-leucine. The addition of cortisol to the incubation media markedly decreased the enhancement of 14C-lysine incorporation into the protein of atrophied adrenals. These, as well as additional results suggest rebound phenomena: once atrophic adrenals are transferred to cortisol-free media, reparative processes begin after a delay period. Such phenomena seem to be mediated by regulatory mechanisms at the adrenal level.

scholarly journals LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i7-i7
Author(s):  
Jiaojiao Deng ◽  
Sophia Chernikova ◽  
Wolf-Nicolas Fischer ◽  
Kerry Koller ◽  
Bernd Jandeleit ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Tumors ◽  
Cell Lines ◽  
Chemotherapeutic Agent ◽  
Leptomeningeal Metastasis ◽  
Breast Cancer Cell Lines ◽  
Normal Brain ◽  
Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Delivery of melittin-loaded niosomes for breast cancer treatment: an in vitro and in vivo evaluation of anti-cancer effect

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Farnaz Dabbagh Moghaddam ◽  
Iman Akbarzadeh ◽  
Ehsan Marzbankia ◽  
Mahsa Farid ◽  
Leila khaledi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Treatment ◽  
Cell Lines ◽  
Encapsulation Efficiency ◽  
Inbred Mice ◽  
Breast Cancer Treatment ◽  
Anticancer Effect ◽  
Anti Cancer

Abstract Background Melittin, a peptide component of honey bee venom, is an appealing candidate for cancer therapy. In the current study, melittin, melittin-loaded niosome, and empty niosome had been optimized and the anticancer effect assessed in vitro on 4T1 and SKBR3 breast cell lines and in vivo on BALB/C inbred mice. "Thin-layer hydration method" was used for preparing the niosomes; different niosomal formulations of melittin were prepared and characterized in terms of morphology, size, polydispersity index, encapsulation efficiency, release kinetics, and stability. A niosome was formulated and loaded with melittin as a promising drug carrier system for chemotherapy of the breast cancer cells. Hemolysis, apoptosis, cell cytotoxicity, invasion and migration of selected concentrations of melittin, and melittin-loaded niosome were evaluated on 4T1 and SKBR3 cells using hemolytic activity assay, flow cytometry, MTT assay, soft agar colony assay, and wound healing assay. Real-time PCR was used to determine the gene expression. 40 BALB/c inbred mice were used; then, the histopathology, P53 immunohistochemical assay and estimate of renal and liver enzyme activity for all groups had been done. Results This study showed melittin-loaded niosome is an excellent substitute in breast cancer treatment due to enhanced targeting, encapsulation efficiency, PDI, and release rate and shows a high anticancer effect on cell lines. The melittin-loaded niosome affects the genes expression by studied cells were higher than other samples; down-regulates the expression of Bcl2, MMP2, and MMP9 genes while they up-regulate the expression of Bax, Caspase3 and Caspase9 genes. They have also enhanced the apoptosis rate and inhibited cell migration, invasion in both cell lines compared to the melittin samples. Results of histopathology showed reduce mitosis index, invasion and pleomorphism in melittin-loaded niosome. Renal and hepatic biomarker activity did not significantly differ in melittin-loaded niosome and melittin compared to healthy control. In immunohistochemistry, P53 expression did not show a significant change in all groups. Conclusions Our study successfully declares that melittin-loaded niosome had more anti-cancer effects than free melittin. This project has demonstrated that niosomes are suitable vesicle carriers for melittin, compare to the free form.

scholarly journals Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Jo Rademacher ◽  
Anahi Cruz ◽  
Mary Faber ◽  
Robyn A. A. Oldham ◽  
Dandan Wang ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Lines ◽  
Nk Cell ◽  
Autologous Tumor ◽  
Murine Models ◽  
Lentiviral Transduction ◽  
Ifn Γ ◽  
Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

Studies of Metabolism Mediated Mutagenicity In Vitro

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 243-250
Author(s):  
Dag Jenssen ◽  
Lennart Romert
Keyword(s):  
Cell Lines ◽  
Biological Effects ◽  
Animal Experiments ◽  
Culture Technique ◽  
Organ Perfusion ◽  
Isolated Cells ◽  
Toxicological Effect ◽  
In Vitro Methods

To understand the cause of the biological effects of xenobiotic metabolism in mammals, investigators have traditionally performed animal experiments by comparing the results of biochemical methods, such as measurement of enzyme activity analysis of the metabolites produced, with the observed toxicological effect. This article deals with in vitro methods for genotoxicity combined with drug metabolising preparations at the organelle, cell or organ levels, as exemplified by microsome preparations, isolated cells/cell lines and organ perfusion systems, respectively. The advantage of some of these methods for studying metabolism-mediated mutagenicity is that the measured endpoint reflects not only the bioactivating phase I reactions, but also the detoxifying phase II reactions, and the transfer of the non-conjugated reactive metabolites to other cells and their ability to cause mutations in these cells. In vivo, all these events are important factors in the initiation of cancer. A mechanistic advantage of the methods for metabolism-mediated mutagenicity in vitro is that the relevance of the different steps in metabolism for the mutational events can seldom be investigated in an in vivo assay. Furthermore, human studies can easily be performed using the co-culture technique with isolated human cells or cell lines.

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