Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines

High interstitial level of ATP and its lysate adenosine in the cancer microenvironment are considered a halo mark of cancer. Adenosine acts as a strong immune suppressor. However, the source of ATP release is unclear. We clarified the release of ATP via volume-regulated anion channels (VRACs) in breast cell lines using an ATP luminescence imaging system. We detected a slowly rising diffuse pattern of ATP release that was only observed in undifferentiated cells, not in differentiated primary cultured cells. This was confirmed by suppression with DCPIB, a blocker of VRACs, and shRNA for LRRC8A, an indispensable subunit of VRACs. We herein demonstrated that the inflammatory mediator sphingosine-1-phosphate (S1P), which exists abundantly in the cancer microenvironment, induced a diffuse pattern of ATP release isovolumetrically. The response was dose-dependent and suppressed by the knock-down of LRRC8A. It was also suppressed by blockers of S1P receptor 1 and 2 (W146 and JTE013, respectively). RTqPCR demonstrated the prominent presence of S1PR1 and S1PR2 mRNAs. We discussed the roles of S1P-induced ATP release in the cancer microenvironment.

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Hypo-osmotic Stress Induces ATP Release via Volume-regulated Anion Channels in Undifferentiated Mammary Cells

10.1101/2021.04.25.441329 ◽

2021 ◽

Author(s):

Kishio Furuya ◽

Yuko Takahashi ◽

Hiroaki Hirata ◽

Takeshi Kobayashi ◽

Mikhail Samsonov ◽

...

Keyword(s):

Cell Lines ◽

Transforming Growth Factor ◽

Cultured Cells ◽

Imaging System ◽

Atp Release ◽

Molecular Entity ◽

Mammary Cells ◽

Anion Channels ◽

Breast Cell Lines ◽

Release Pattern

The high interstitial ATP concentration in the cancer microenvironment is a major source of adenosine, which acts as a strong immune suppressor. However, the source of ATP release has not been elucidated. We measured the ATP release during hypotonic stress using a real-time ATP luminescence imaging system in primary cultured mammary cells and in breast cell lines. In primary cultured cells, ATP was intermittently released with transient-sharp peaks, while in breast cell lines ATP was released with a slowly rising diffuse pattern. The diffuse ATP release pattern was changed to a transient-sharp pattern by cholera toxin treatment and the reverse change was induced by transforming growth factor (TGF) β treatment. DCPIB, an inhibitor of volume-regulated anion channels (VRACs), only suppressed the diffuse pattern. The inflammatory mediator sphingosine-1-phosphate (S1P) induced a diffuse ATP release pattern isovolumetrically. The knockdown of A isoform of leucine-rich repeat-containing protein 8 (LRRC8A), the essential molecular entity of VRACs, using shRNA suppressed the diffuse pattern. These results suggest that abundantly expressed VRACs are a conduit of ATP release in undifferentiated cells, including cancer cells.

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Sphingolipid Pathway as a Source of Vulnerability in IDH1mut Glioma

Cancers ◽

10.3390/cancers12102910 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2910 ◽

Cited By ~ 1

Author(s):

Tyrone Dowdy ◽

Lumin Zhang ◽

Orieta Celiku ◽

Sriya Movva ◽

Adrian Lita ◽

...

Keyword(s):

Cell Lines ◽

Glioma Cell ◽

Model Systems ◽

Sphingolipid Metabolism ◽

Small Scale ◽

Rational Drug ◽

Glioma Cell Lines ◽

Sphingosine 1 Phosphate ◽

Tumor Phenotype ◽

Dose Dependent

In addition to providing integrity to cellular structure, the various classes of lipids participate in a multitude of functions including secondary messengers, receptor stimulation, lymphocyte trafficking, inflammation, angiogenesis, cell migration, proliferation, necrosis and apoptosis, thus highlighting the importance of understanding their role in the tumor phenotype. In the context of IDH1mut glioma, investigations focused on metabolic alterations involving lipidomics’ present potential to uncover novel vulnerabilities. Herein, a detailed lipidomic analysis of the sphingolipid metabolism was conducted in patient-derived IDH1mut glioma cell lines, as well as model systems, with the of identifying points of metabolic vulnerability. We probed the effect of decreasing D-2HG levels on the sphingolipid pathway, by treating these cell lines with an IDH1mut inhibitor, AGI5198. The results revealed that N,N-dimethylsphingosine (NDMS), sphingosine C17 and sphinganine C18 were significantly downregulated, while sphingosine-1-phosphate (S1P) was significantly upregulated in glioma cultures following suppression of IDH1mut activity. We exploited the pathway using a small-scale, rational drug screen and identified a combination that was lethal to IDHmut cells. Our work revealed that further addition of N,N-dimethylsphingosine in combination with sphingosine C17 triggered a dose-dependent biostatic and apoptotic response in a panel of IDH1mut glioma cell lines specifically, while it had little effect on the IDHWT cells probed here. To our knowledge, this is the first study that shows how altering the sphingolipid pathway in IDH1mut gliomas elucidates susceptibility that can arrest proliferation and initiate subsequent cellular death.

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Prolonged Survival of Chronic Lymphocytic Leukemia Cells Induced by Sphingosine 1-Phosphate Is Mediated by the G Protein-Coupled Receptor S1P1 and Involves Erk/MAP Kinase, but Not Akt Signaling.

Blood ◽

10.1182/blood.v108.11.2801.2801 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2801-2801

Author(s):

Gabriele Seitz ◽

Sedat Yildirim ◽

Andreas Boehmler ◽

Lothar Kanz ◽

Robert Mohle

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Map Kinase ◽

Mrna Expression ◽

Cell Lines ◽

Lymphocytic Leukemia ◽

Receptor Internalization ◽

Prolonged Survival ◽

S1p1 Receptor ◽

Sphingosine 1 Phosphate ◽

Dose Dependent

Abstract Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator which is generated by the hydrolysis of sphingomyelin and released predominantely by activated platelets but also by other cells upon activation. In normal blood plasma it is found in nanomolar to micromolar concentrations. By qualitative and quantitative (TaqMan) RT-PCR, mRNA expression of the S1P receptor S1P1 was detected in CLL cell lines (EHEB, MEC-1) and in all samples of primary chronic lymphocytic leukemia (B-CLL) cells, analyzed after isolation with a CD19 antibody and magnetic cell sorting. mRNA expression of other S1P receptors was less consistently found. When primary CLL cells were cultured in serum-free medium (RPMI 1640 supplemented with fatty acid-free BSA), viability decreased starting from 3 days of culture and was completely lost after 1–2 weeks depending on the individual sample. However, when the cultures were supplemented with S1P (1 nM - 5 μM) the number of viable cells was increased up to 8-fold in a time- and dose-dependent manner as measured using the WST-1 reagent. Moreover, S1P induced a rapid (maximum at 1 min) phosphorylation of the p44/42 Erk/MAP kinase in CLL cell lines and even more prominent in primary CLL cells. The time- and dose dependent phosphorylation was blocked by pretreatment (24h) of the cells with FTY720 (10–100 nM), a compound that binds predominantly to S1P1 and induces loss of cell surface expression by receptor internalization. FTY720 also abrogated the prolonged survival of CLL cells in cultures supplemented with S1P, demonstrating that both the Erk/MAP kinase phosphorylation and the consecutive increased survival were mediated by the S1P1 receptor. Activation of MAP kinase was also inhibited by pertussis toxin, which selectively blocks Gi proteins, the only class of G proteins known to be coupled to S1P1. Phosphorylation of Akt was not observed in CLL cell lines and primary CLL cells after stimulation with S1P. B-CLL cell lines showed a pronounced chemotactic response to S1P at low concentrations. In primary CLL cells however, S1P did neither induce phosphorylation of the focal adhesion kinase (FAK) nor chemotaxis. We conclude that S1P, constitutively present in the blood, prolongs survival of CLL cells involving the Erk/MAP kinase pathway. Since compounds that induce S1P1 receptor internalization like FTY720 are already used as immunosuppressants in humans, our results suggest inclusion of these drugs also in the treatment of B-CLL, where they may potentiate the effect of conventional antiproliferative therapy.

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Antioxidant effects and in vitro cytotoxicity on human cancer cell lines of flavonoid-rich Flamboyant (Delonix regia (Bojer) Raf.) flower extract

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666201029154746 ◽

2020 ◽

Vol 21 ◽

Author(s):

Putthiporn Khongkaew ◽

Phanphen Wattanaarsakit ◽

Konstantinos I. Papadopoulos ◽

Watcharaphong Chaemsawang

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Flower Extract ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Murine Leukemia Cell ◽

Dose Dependent

Background: Cancer is a noncommunicable disease with increasing incidence and mortality rates both worldwide and in Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Introduction: Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. Methods and Results: In the present report, using High Performance Liquid Chromatography (HPLC), we demonstrate in the DRE, the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30 % w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01 % w/w respectively while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 µg/ml and 53.65±7.24 µg/ml respectively. Discussion: Our cancer cell line studies using the MTT assay demonstrated DREs potent and dose dependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Conclusion: Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract’s lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

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Synthesis, Spectral Characterization, Antibacterial and Anticancer Activity of some Titanium Complexes

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666171219122431 ◽

2018 ◽

Vol 18 (5) ◽

pp. 739-746 ◽

Cited By ~ 2

Author(s):

Raj Kaushal ◽

Nitesh Kumar ◽

Archana Thakur ◽

Kiran Nehra ◽

Pamita Awasthi ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Anticancer Activity ◽

Cell Lines ◽

Spectral Characterization ◽

Mechanistic Study ◽

Titanium Complexes ◽

G0 Phase ◽

Antibacterial And Anticancer Activity ◽

Dose Dependent

Abstract: Background: After the discovery of cisplatin, first non platinum anticancer drugs having excellent efficacy were budotitane and TiCl2(cp)2 but action mechanism is not clear. Therefore, we hereby reporting synthesis and biological activities novel titanium complexes to explore their mode of action. Objectives: Synthesis, spectral characterization, antibacterial and anticancer activity of some titanium complexes. Antibacterial studies on various bacterial strains and anticancer studies on HeLa, C6, CHO cancerous cell lines have been performed. Further, the cell death mechanistic study was done on CHO cell lines. Method: Titanium complexes with and without labile groups have been synthesized by reacting of TiCl4 with nitrogen containing ligands viz. 1,2-diaminocyclohexane, 1,10-Phenanthroline, adamantylamine, 2,2'-bipyridine, 4,4'-dimethyl-2,2'-bipyridine in predetermined molar ratios. Antibacterial and anticancer studies were performed by agar well diffusion method and MTT assay respectively. Cell cycle analysis is done by using flow cytometry. Results: Complex 2 i.e TiCl2(Phen)2 showed better activity than other complexes as an antibacterial as well as anticancer agent. Phase contrast imaging indicates that observed morphological changes of cells was dose dependent. Cell death mechanistic study have shown the increase in sub G0 phase population as well as formation of blebbing and fragmentation of chromatin material which is an indicative measure of apoptosis. Conclusion: Complex 2 proved to be more effective bactericide and cytotoxic agent. Cell cycle analysis showed cell arrest in G0 phase. Apoptosis percentage was found to increase in a dose dependent manner. So, prepared titanium complexes can be put to use as an important chemotherapeutic agents.

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PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

Applied Sciences ◽

10.3390/app11093729 ◽

2021 ◽

Vol 11 (9) ◽

pp. 3729

Author(s):

Katarzyna Balon ◽

Benita Wiatrak

Keyword(s):

Cell Culture ◽

Dna Damage ◽

Cell Lines ◽

Inflammatory Factor ◽

Research Model ◽

Differentiated Cells ◽

Undifferentiated Cells ◽

Primary Cell Lines ◽

Neurobiological Research ◽

The Impact

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

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Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

Alternatives to Laboratory Animals ◽

10.1177/026119298801600108 ◽

1988 ◽

Vol 16 (1) ◽

pp. 32-37

Author(s):

Margherita Ferro ◽

Anna Maria Bassi ◽

Giorgio Nanni

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Membrane Lipids ◽

Hepatoma Cell ◽

Positive Control ◽

In Vitro Models ◽

Low Concentrations ◽

Formation Efficiency ◽

Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

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Direct protein delivery into intact plant cells using polyhistidine peptides

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab055 ◽

2021 ◽

Author(s):

Yoshino Tanaka ◽

Yoshihiko Nanasato ◽

Kousei Omura ◽

Keita Endoh ◽

Tsuyoshi Kawano ◽

...

Keyword(s):

Cell Lines ◽

Fusion Proteins ◽

Protein Delivery ◽

Fluorescent Protein ◽

Cultured Cells ◽

Plant Cells ◽

Adenosine Monophosphate ◽

Cell Penetrating Peptides ◽

Intracellular Space ◽

Intracellular Fluorescence

Abstract Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

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Cytocompatibility of stabilized black phosphorus nanosheets tailored by directly conjugated polymeric micelles for human breast cancer therapy

Scientific Reports ◽

10.1038/s41598-021-88791-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

M. Biedulska ◽

P. Jakóbczyk ◽

M. Sosnowska ◽

B. Dec ◽

A. Muchlińska ◽

...

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Polymeric Micelles ◽

Black Phosphorus ◽

Environmental Stability ◽

Breast Cancer Cell Lines ◽

The Novel ◽

Ft Ir ◽

Dose Dependent ◽

Aggressive Breast Cancer

AbstractThe novel procedure of few-layer black phosphorus (FLBP) stabilization and functionalisation was here proposed. The cationic polymer PLL and non-ionic PEG have been involved into encapsulation of FLBP to allow sufficient time for further nanofabrication process and overcome environmental degradation. Two different spacer chemistry was designed to bind polymers to tumor-homing peptides. The efficiency of functionalisation was examined by RP-HPLC, microscopic (TEM and SEM) and spectroscopic (FT-IR and Raman) techniques as well supported by ab-initio modelling. The cell and dose dependent cytotoxicity of FLBP and its bioconjugates was evaluated against HB2, MCF-7 and MDA-MB-231 cell lines. Functionalisation allowed not only for improvement of environmental stability, but also enhances therapeutic effect by abolished the cytotoxicity of FLBP against HB2 cell line. Moreover, modification of FLBP with PLL caused increase of selectivity against highly aggressive breast cancer cell lines. Results indicate the future prospect application of black phosphorus nanosheets as nanocarrier, considering its unique features synergistically with conjugated polymeric micelles.

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Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Scientific Reports ◽

10.1038/s41598-021-84551-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alessandro T. Caputo ◽

Oliver M. Eder ◽

Hana Bereznakova ◽

Heleen Pothuis ◽

Albert Ardevol ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cell ◽

Recombinant Proteins ◽

Cultured Cells ◽

Hek293 Cells ◽

Reaction Products ◽

Enzyme Form ◽

X Ray Crystallography ◽

Mammalian Cell Lines ◽

Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

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