scholarly journals Detection of glucose-derived d- and l-lactate in cancer cells by the use of a chiral NMR shift reagent

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Eul Hyun Suh ◽  
Carlos F. G. C. Geraldes ◽  
Sara Chirayil ◽  
Brandon Faubert ◽  
Raul Ayala ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Red Blood Cells ◽  
Cancer Cells ◽  
Cell Lines ◽  
Blood Cells ◽  
Lactate Production ◽  
Shift Reagent ◽  
Nsclc Cell ◽  
Parental Cell Line ◽  
H Nmr

Abstract Background Excessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to l-lactate. Although d-lactate can also be produced from glucose via the methylglyoxal pathway in small amounts, less is known about the amount of d-lactate produced in cancer cells. Since the stereoisomers of lactate cannot be distinguished by conventional 1H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1H NMR resonances of d- and l-lactate. Methods The production of l-lactate from glucose and d-lactate from methylglyoxal was first demonstrated in freshly isolated red blood cells using the chiral NMR shift reagent, YbDO3A-trisamide. Then, two different cell lines with high GLO1 expression (H1648 and H 1395) were selected from a panel of over 80 well-characterized human NSCLC cell lines, grown to confluence in standard tissue culture media, washed with phosphate-buffered saline, and exposed to glucose in a buffer for 4 h. After 4 h, a small volume of extracellular fluid was collected and mixed with YbDO3A-trisamide for analysis by 1H NMR spectroscopy. Results A suspension of freshly isolated red blood cells exposed to 5mM glucose produced l-lactate as expected but very little d-lactate. To evaluate the utility of the chiral NMR shift reagent, methylglyoxal was then added to red cells along with glucose to stimulate the production of d-lactate via the glyoxalate pathway. In this case, both d-lactate and l-lactate were produced and their NMR chemical shifts assigned. NSCLC cell lines with different expression levels of GLO1 produced both l- and d-lactate after incubation with glucose and glutamine alone. A GLO1-deleted parental cell line (3553T3) showed no production of d-lactate from glucose while re-expression of GLO1 resulted in higher production of d-lactate. Conclusions The shift-reagent-aided NMR technique demonstrates that d-lactate is produced from glucose in NSCLC cells via the methylglyoxal pathway. The biological role of d-lactate is uncertain but a convenient method for monitoring d-lactate production could provide new insights into the biological roles of d- versus l-lactate in cancer metabolism.

Cytotoxicity of chitosan ultrafine nanoshuttles on MCF-7 cell line as surrogate model for breast cancer

2020 ◽  
Vol 17 ◽  
Author(s):  
Tarek Faris ◽  
Gamaleldin I. Harisa ◽  
Fars K. Alanazi ◽  
Mohamed M. Badran ◽  
Afraa Mohammad Alotaibi ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Factorial Design ◽  
Cell Line ◽  
Cell Lines ◽  
Blood Cells ◽  
Sodium Tripolyphosphate ◽  
Physicochemical Characterization ◽  
Spherical Shape ◽  
Mcf 7

Aim: This study aimed to explore an affordable technique for the fabrication of Chitosan Nanoshuttles (CSNS) at the ultrafine nanoscale less than 100 nm with improved physicochemical properties, and cytotoxicity on the MCF-7 cell line. Background: Despite several studies reported that the antitumor effect of CS and CSNS could achieve intracellular compartment target ability, no enough available about this issue and further studies are required to address this assumption. Objectives: The objective of the current study was to investigate the potential processing variables for the production of ultrafine CSNS (> 100 nm) using Box-Benhken Design factorial design (BBD). This was achieved through a study of the effects of processing factors, such as CS concentration, CS/TPP ratio, and pH of the CS solution, on PS, PDI, and ZP. Moreover, the obtained CSNS was evaluated for physicochemical characteristics, morphology Also, hemocompatibility, and cytotoxicity using Red Blood Cells (RBCs) and MCF-7 cell lines were investigated. Methods: Box-Benhken Design factorial design (BBD) was used in the analysis of different selected variables. The effects of CS concentration, sodium tripolyphosphate (TPP) ratio, and pH on particle size, Polydispersity Index (PDI), and Zeta Potential (ZP) were measured. Subsequently, the prepared CS nanoshuttles were exposed to stability studies, physicochemical characterization, hemocompatibility, and cytotoxicity using red blood cells and MCF-7 cell lines as surrogate models for in vivo study. Result: The present results revealed that the optimized CSNS have ultrafine nanosize, (78.3±0.22 nm), homogenous with PDI (0.131±0.11), and ZP (31.9±0.25 mV). Moreover, CSNS have a spherical shape, amorphous in structure, and physically stable. Also, CSNS has biological safety as indicated by a gentle effect on red blood cell hemolysis, besides, the obtained nanoshuttles decrease MCF-7 viability. Conclusion: The present findings concluded that the developed ultrafine CSNS has unique properties with enhanced cytotoxicity. thus promising for use in intracellular organelles drug delivery.

scholarly journals Interaction of Arylidenechromanone/Flavanone Derivatives with Biological Macromolecules Studied as Human Serum Albumin Binding, Cytotoxic Effect, Biocompatibility Towards Red Blood Cells

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3172 ◽  
Author(s):  
Angelika A. Adamus-Grabicka ◽  
Magdalena Markowicz-Piasecka ◽  
Michał B. Ponczek ◽  
Joachim Kusz ◽  
Magdalena Małecka ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Red Blood Cells ◽  
Human Serum ◽  
Cell Line ◽  
Cell Lines ◽  
Blood Cells ◽  
Cytotoxic Effect ◽  
Human Leukemia ◽  
Ic50 Values

The aim of this study was to determine the cytotoxic effect of 3-arylidenechromanone (1) and 3arylideneflavanone (2) on HL-60 and NALM-6 cell lines (two human leukemia cell lines) and a WM-115 melanoma cell line. Both compounds exhibited high cytotoxic activity with higher cytotoxicity exerted by compound 2, for which IC50 values below 10 µM were found for each cell line. For compound 1, the IC50 values were higher than 10 µM for HL-60 and WM-115 cell lines, but IC50 < 10 µM was found for the NALM-6 cell line. Both compounds, at the concentrations close to IC50 (concentration range: 5–24 µM/L for compound 1 and 6–10 µM/L for compound 2), are not toxic towards red blood cells. The synthesized compounds were characterized using spectroscopic methods 1H- and 13C-NMR, IR, MS, elemental analysis, and X-ray diffraction. The lipophilicity of both synthesized compounds was determined using an RP-TLC method and the logP values found were compared with the theoretical ones taken from the Molinspiration Cheminformatics (miLogP) software package. The mode of binding of both compounds to human serum albumin was assessed using molecular docking methods.

scholarly journals Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin

2020 ◽  
Vol 21 (5) ◽  
pp. 1661
Author(s):  
Anamarija Mojzeš ◽  
Marko Tomljanović ◽  
Lidija Milković ◽  
Renata Novak Kujundžić ◽  
Ana Čipak Gašparović ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Warburg Effect ◽  
Aerobic Glycolysis ◽  
Intracellular Localization ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Cell Type ◽  
Cell Type Specific ◽  
The Warburg Effect

In order to support uncontrolled proliferation, cancer cells need to adapt to increased energetic and biosynthetic requirements. One such adjustment is aerobic glycolysis or the Warburg effect. It is characterized by increased glucose uptake and lactate production. Curcumin, a natural compound, has been shown to interact with multiple molecules and signaling pathways in cancer cells, including those relevant for cell metabolism. The effect of curcumin and its solvent, ethanol, was explored on four different cancer cell lines, in which the Warburg effect varied. Vital cellular parameters (proliferation, viability) were measured along with the glucose consumption and lactate production. The transcripts of pyruvate kinase 1 and 2 (PKM1, PKM2), serine hydroxymethyltransferase 2 (SHMT2) and phosphoglycerate dehydrogenase (PHGDH) were quantified with RT-qPCR. The amount and intracellular localization of PKM1, PKM2 and signal transducer and activator of transcription 3 (STAT3) proteins were analyzed by Western blot. The response to ethanol and curcumin seemed to be cell-type specific, with respect to all parameters analyzed. High sensitivity to curcumin was present in the cell lines originating from head and neck squamous cell carcinomas: FaDu, Detroit 562 and, especially, Cal27. Very low sensitivity was observed in the colon adenocarcinoma-originating HT-29 cell line, which retained, after exposure to curcumin, a higher levels of lactate production despite decreased glucose consumption. The effects of ethanol were significant.

Monocarboxylate Transporters Play an Important Role in Aerobicglycolysis in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1821-1821
Author(s):  
Shiho Fujiwara ◽  
Naoko Wada ◽  
Yawara Kawano ◽  
Hiromichi Yuki ◽  
Yutaka Okuno ◽  
...  
Keyword(s):  
Energy Source ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cancer Cells ◽  
Cell Lines ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Monocarboxylate Transporters ◽  
Dependent Manner ◽  
Growth And Survival

Abstract Abstract 1821 Introduction It has been reported that cancer cells utilize glycolysis pathway (non-oxidative breakdown of glucose) even in the presence of adequate oxygen to provide cancer cells with energy, called the Warburg effect (aerobic glycolysis) that ultimately leads to produce lactate. We reported in the last ASH meeting that aerobic glycolysis is up-regulated in multiple myeloma (MM) cells in patients with high serum LDH levels and aerobic glycolysis itself could serve as a novel therapeutic target in MM patients. Here we report an importance of lactate transporter for the growth and survival of MM cells. Lactate, produced from pyruvate by lactate dehydrogenase A (LDHA), is known as an important energy source for solid tumor cells and is associated with tumor angiogenesis and chemo-resistance (Pinheiro, C., et al. J Bioenerg Biomembr. 44:127–139, 2012). On the other hand, LDHB converts lactate to pyruvate, thus negatively regulating lactate production. It is known that lactate is pumped out through monocarboxylate trasnporter, MCT4, while MCT1 mainly imports lactate to inside of cells. However, roles of MCT1 and MCT4 in MM cells remain to be elucidated. We here investigated the roles of these two molecules in the growth and survival of MM cells. CD147, a purported chaperone protein for MCT1, was also examined. Methods Six MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were employed. Six genes associated with glycolysis, i.e., LDHA, LDHB, MCT1-4, were examined using real time PCR analysis. Expressions of MCT1 and MCT4 were analyzed with western blotting. Expression of CD147 was investigated by flow cytometry. Lactate production into culture supernatants of MM cell lines were analyzed by using a lactate analyzer. An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells. AnnexinV/PI stained cells was analyzed by flow cytometry to quantify cytotoxicity. MCT1-expression was inhibited by using siRNA. Dichroloacetate (DCA), an inhibitor of PDK1, was utilized for inhibiting glycolysis. Results Accumulation of lactate was found in the supernatants of MM cell lines as cell density increased. Transporters of lactate, MCT1, MCT4 and CD147, were found in most MM cell lines at various levels, suggesting that transportation of lactate occurs through membrane of MM cells. To examine the role of lactate as a growth promotion factor, lactate was exogenously supplemented to KMS-12-PE cells. Interestingly, expressions of MCT1 and LDHB genes increased by the addition of lactate while those of MCT4 and LDHA only moderately changed (Fig. 1), suggesting that lactate was imported to cells through MCT1, then converted to pyrvate by LDHB. These results raised a possibility that lactate is utilized by MM cells as a growth factor. To examine the possibility, CHC, an inhibitor of MCT1, was supplemented to MM cell cultures. Interestingly, CHC induced apoptosis in MM cells in a dose dependent manner (Fig. 2). Moreover, inhibition of MCT1 gene by siRNA showed significant induction of apoptosis (Fig. 3), strongly suggesting that MCT1 plays a crucial role for survival of MM cells. Finally, we found a significant increase in the apoptosis of MM cells when CHC and DCA were simultaneously added in the culture (Fig.4), suggesting that MCT1 functions independently from glycolysis per se and that CHC and DCA act additively in starving lactate within MM cells. Conclusion Our results suggest that lactate is actively transported through monocarboxylate transporters. Given the results that exogenous lactate production increased MCT1 and LDHB expression, lactate should play a role as a regulator of lactate transportation and glycolysis as well as an important energy source. Because we found significant amount of lactate was produced from stromal cells obtained from MM patients, lactate may be supplied not only from MM cells themselves but also from micro-environment. Our finding that inhibition of MCT1 leads to cell death suggests that MCT1 could be a potential novel target molecule in MM therapy that could be stratified in combination with glycolysis inhibitor. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Erythroblasts with c-Kit Activating Mutations Have Reduced Cell Culture Costs and Remain Capable of Terminal Maturation and Enucleation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2315-2315
Author(s):  
Tyler A Couch ◽  
Zachary C. Murphy ◽  
Michael Getman ◽  
Ryo Kurita ◽  
Yukio Nakamura ◽  
...  
Keyword(s):  
Cell Culture ◽  
Red Blood Cells ◽  
Cell Lines ◽  
Blood Cells ◽  
In Vitro Production ◽  
Activating Mutations ◽  
Expansion Medium ◽  
The Cost ◽  
Vitro Production

Abstract There is a constant need for red blood cells for transfusion therapy in the treatment of anemias and acute injury. As all blood products for transfusion come from donors, there are concerns over shortages and safety. Furthermore, many patients with transfusion-dependent anemias risk alloiumminization. The in vitro production of red blood cells would address these problems, especially as they can be genetically engineered to prevent alloimmunization. Numerous erythroid culture systems now exist for the in vitro production of red blood cells. Hematopoietic stem and progenitor cells (HSPCs) obtained from umbilical cord or peripheral blood can be differentiated into erythrocytes, however, they are limited in expansion. While umbilical cord HSPCs have greater expandability than peripheral blood, the resulting erythrocytes contain fetal globins. Pluripotent stem cells can also be used as a starting source, however only a small percentage of the cells can be differentiated into erythroblasts which also suffer from low enucleation rates. Presently, the cost of in vitro production of a unit of red cells is greater than an order of magnitude higher than obtaining it from a donor largely due to the medium and cytokine costs (Timmins & Nielsen, Trends Biotechnol, 2009). A relatively new approach of immortalizing early erythroblasts allowing unlimited expansion as well as terminal maturation and enucleation shows great therapeutic promise (Kurita et al., PLoS One, 2013; Huang et al., Mol Ther, 2014; Trakarnsanga et al., Nat Commun, 2017). However, these immortalized erythroblasts are still reliant on two costly cytokines: stem cell factor (SCF) and erythropoietin (Epo). Mutations in exon 17 of the receptor tyrosine kinase gene KIT are frequently seen in acute myeloid leukemias, gastrointestinal stromal tumors, and mast cells leading to mastocytosis. These mutations cause the c-Kit protein to spontaneously activate and transduce signal in the absence of SCF (Kit-ligand). To generate an SCF-independent HUDEP-2 cell line (Kurita et al., PLoS One, 2013), we used CRISPR/Cas9 to introduce missense and frameshifting mutations within the vicinity of Asp816 in exon 17 of the KIT gene. The resulting monoclonal cell lines were selected for by removing SCF from the expansion medium and were subsequently named KIT-CAT (KIT with Constitutively Activating Transformation). To better understand what KIT mutations allowed or impaired terminal maturation, monoclonal cell lines were genotyped by Sanger sequencing. Three cell lines with unique genotypes were chosen for further analysis. All three KIT-CAT lines had a shorter doubling time compared to HUDEP-2 cells (16.7 vs 18.9 hrs, p=0.020) and were no longer dependent on SCF or Epo. However, two of the three KIT-CAT lines showed more robust proliferation with Epo in the expansion medium. The addition of SCF to the medium caused no increase in c-Kit activation by Western blotting for phosphorylation at Tyr703. Furthermore, the low molecular weight and immature form of c-Kit is also phosphorylated in KIT-CAT cells, but not HUDEP-2 cells, indicating c-Kit activation occurs before trafficking to the cell membrane where SCF would bind (Tabone-Eglinger et al., Clin Cancer Res, 2008). Key features of erythroblast maturation are the decrease in cell and nuclear size which can be measured using imaging flow cytometry (McGrath et al., Methods, 2017). While in expansion phase, all 3 cell lines were larger in cell and nuclear area compared to the parental HUDEP-2 line. By day 6 of maturation, all three cell lines had statistically significant decreases in cell and nuclear size indicating maturation. By day 13 of culture, Wright-Giemsa staining showed that the majority of the cells were orthochromatic erythroblasts or enucleate reticulocytes. Reducing cell culture costs is needed for in vitro manufacturing of red blood cells to be economically feasible. These results show that a c-Kit activating mutations in human erythroblasts removes the cost of SCF and reduces the cost of Epo while still allowing for terminal maturation and enucleation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of gadolinium-based magnetic resonance imaging contrast media on red blood cells and K562 cancer cells

2020 ◽  
Vol 62 ◽  
pp. 126640
Author(s):  
Benjamaporn Supawat ◽  
Phattharawadi Moungthong ◽  
Chananchida Chanloi ◽  
Natchaporn Jindachai ◽  
Singkome Tima ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Magnetic Resonance ◽  
Contrast Media ◽  
Red Blood Cells ◽  
Cancer Cells ◽  
Blood Cells ◽  
Resonance Imaging ◽  
Magnetic Resonance Imaging Contrast

Methionine gamma-lyase-encapsulated into red blood cells (ERY-MET) antitumor activity in gastric carcinoma.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 78-78
Author(s):  
Vanessa Bourgeaux ◽  
Karine Sénéchal ◽  
Karine Aguera ◽  
Fabien Gay ◽  
Françoise Horand
Keyword(s):  
Gastric Carcinoma ◽  
Tumor Growth ◽  
Red Blood Cells ◽  
Cell Lines ◽  
Blood Cells ◽  
Half Life ◽  
Anti Tumor Activity ◽  
Methionine Gamma Lyase

78 Background: Methionine (Met) requirement is a cancer specific–metabolic defect that seems a promising target, especially in gastric cancers. Methionine gamma–lyase (MGL), a pyridoxal–5′–phosphate (PLP)–dependent enzyme, is an emerging approach consisting in tumors Met starvation via systemic Met depletion. ERY-MET is a new therapeutic product overcoming the short in vivo half-life of free MGL by its encapsulation into Red Blood Cells (RBCs). Indeed, ERY-MET works as a bioreactor degrading Met that passively diffuses inside the RBC. In addition, entrapped MGL activity can be controlled by supplying Vitamin B6 (PN), the precursor of MGL’s cofactor (PLP), converted inside RBCs. ERY-MET anti-tumor activity was evaluated in vivo in NMRI nudemice bearing subcutaneous gastric carcinoma. Methods: First, in vitro sensitivity of NCI-N87 and AGS human gastric cell lines to free MGL was assessed by IC50 determination using CCK–8 assay. MGL encapsulated into mouse RBCs by hypotonic dialysis was injected once in CD1 mice to determine PK-PD parameters with or without PN supplementation. The anti-tumor activity of weekly ERY-MET injections (x5) at 116 IU/kg ± 25% was assessed with or without PN supplementation in female NMRI nudemice (n = 10/group) xenografted with NCI-N87 cells. Met depletion was determined 6 days after each cumulative injection while tumor growth was followed twice a week by caliper measurement. Results: In vitro studies showed that NCI-N87 as well as AGS cell lines displayed a sensitivity to free MGL with IC50 of 0.35 ± 0.01 and 0.12 ± 0.02 IU/mL, respectively. ERY-MET with daily PN supplementation significantly increased active MGL half-life in vivo (from < 24h to 8–9 days). ERY-MET induced 80% inhibition of tumor growth at day 45 (p < 0.0001). Response rate obtained was 76% of treated mice (15/20). Besides, PN supplementation induced a slow-down of tumor growth during the supplementation period and improved ERY-MET efficacy. Conclusions: Theses results suggest that ERY-MET can induce tumor growth inhibition in mice bearing human gastric adenocarcinoma and that its effect can be regulated by PN supplementation. As such, ERY-MET seems a promising anti-tumor drug to treat gastric cancers.

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