Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun;56(2):128-137.
doi: 10.1007/s00795-022-00347-4. Epub 2023 Jan 9.

Gemcitabine alters sialic acid binding of the glycocalyx and induces inflammatory cytokine production in cultured endothelial cells

Mariko Gunji  1 , Chika Sawa  1 , Minako Akiyama  1 , Shumpei Mukai  2 , Takashi Takaki  1   3 , Dedong Kang  1 , Kazuho Honda  4
Affiliations

Gemcitabine alters sialic acid binding of the glycocalyx and induces inflammatory cytokine production in cultured endothelial cells

Mariko Gunji et al. Med Mol Morphol. 2023 Jun.

Abstract

Gemcitabine (GEM) is an anticancer drug inhibiting DNA synthesis. Glomerular thrombotic microangiopathy (TMA) has been reported as an adverse effect. However, the precise mechanism of GEM-induced endothelial injury remains unknown. Cultured human umbilical vein endothelial cells (HUVECs) in the confluent phase were exposed to GEM (5-100 μM) for 48 h and evaluated cell viability and morphology, lectin binding concerning sialic acid of endothelial glycocalyx (GCX), and immunofluorescent staining of platelet-endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor receptor 2 (VEGFR2). The mRNA expression of α2,6-sialyltransferase (ST6Gal1), sialidase (neuraminidase-1: NEU-1), and interleukin (IL)-1β and IL-6 was also evaluated. GEM exposure at 5 μM induced cellular shrinkage and intercellular dissociation, accompanied by slight attenuation of PECAM and VEGFR2 immunostaining, although cell viability was still preserved. At this concentration, lectin binding showed a reduction of terminal sialic acids in endothelial GCX, probably associated with reduced ST6Gal1 mRNA expression. IL-1β and IL-6 mRNA expression was significantly increased after GEM exposure. GEM reduced terminal sialic acids in endothelial GCX through mRNA suppression of ST6Gal1 and induced inflammatory cytokine production in HUVECs. This phenomenon could be associated with the mechanism of GEM-induced TMA.

Keywords: Endothelial cell; Gemcitabine; Glycocalyx; Interleukin-1β; Interleukin-6; Platelet–endothelial cell adhesion molecule (PECAM); Sialic acid; Vascular endothelial growth factor receptor 2 (VEGFR2).

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest in any components of this study and the manuscript.

Figures

Fig. 1
Fig. 1
Structure and components of human endothelial glycocalyx (GCX). GCX is present on the cell surface of endothelial cells. Glycosaminoglycans (GAGs) such as hyaluronic acid, heparan sulfate, and chondroitin sulfate are attached to the core proteins of GCX. In humans, sialic acid (Neu5AAC) is terminally bound to α-2,6-galactose (Gal) of the sugar chains of these GAGs. Neu5Ac, N-acetyl-neuraminic acid; Gal, galactose; GlcNAc, N-acetyl-D-glucosamine; Man, mannose
Fig. 2
Fig. 2
Effect of GEM on cell viability of HUVECs in growth and confluent phases. a Cell viability assay in the growth phase. GEM in various concentrations ranging from 0.025 to 0.5 μM was added to the culture medium on the first day after cell seeding, and an MTT assay was conducted on the third day (48 h later). The half-maximal (50%) inhibitory concentration (IC50) of cell viability was 0.025–0.05 μM in the growth phase. b Cell viability assay in the confluent phase. GEM at various concentrations ranging from 0.05 to 100 μM was added to the culture medium on the fifth day after cell seeding, and an MTT assay was conducted on the seventh day (48 h later). The IC50 of cell viability was 50–100 μM in the confluent phase. The GEM sensitivity of HUVECs was approximately 1/1,000 to 1/4,000 in the confluent phase compared to the growth phase
Fig. 3
Fig. 3
Cellular morphology and PECAM (CD31) and VEGFR expressions in HUVECs after GEM exposure. a Cellular morphology of HUVECs after GEM exposure in the confluent phase. The cellular morphology showed no significant alterations at 5 μM but began showing a tendency of oval- or spindle-shaped shrinkage and intercellular widening at 50 μM; this tendency became evident in 100 μM of GEM concentration. (× 20). Scale bars: 100 μm. b Immunofluorescent expression of PECAM in HUVECs after GEM exposure CD31. PECAM is expressed onto the plasma membrane in the control state. GEM exposure at 5 µM slightly attenuated the PECAM expression, and it became weaker at 50 µM. The lining of the positivity on the cell membrane became irregular and disrupted at 100 µM [Red: PECAM, Blue: DAPI (nuclei)]. Scale bars: 100 μm. c Immunofluorescent expression of VEGFR2 in HUVECs after GEM exposure. VEGFR2 is mainly present in the cytoplasm in the control state, and it slightly attenuated after GEM exposure at 5 µM and became more scattered as the concentration of GEM increased from 50 µM to 100 µM. [Green: VEGFR2, Blue: DAPI (nucleus)]. Scale bars: 100 μm. d PECAM and VEGFR2 mRNA expression in HUVECs after GEM exposure (RT-PCR). PECAM and VEGFR2 mRNA expression were both detected in HUVECs by PT-PCR. After GEM exposure, the PECAM mRNA expression did not remarkably change at 5 μM but decreased at 50 μM and 100 μM. The VEGRF2 mRNA expression also did not remarkably change at 5 μM but reduced at 50 μM and 100 μM
Fig. 4
Fig. 4
Lectin binding and sialic acid-related enzyme expression in HUVECs after GEM exposure. a Lectin binding of endothelial GCX in HUVECs after GEM exposure. WGA: Exposure to GEM at 5, 50, and 100 μM concentrations did not change WGA-lectin binding notably compared with the control state. SNA: SNA lectin binding was slightly reduced in 5 μM and further diminished in 50 μM and 100 μM. RCA-I: RCA-I binding was sparsely present on the cell membrane in the control state and significantly increased after GEM exposure at 5 μM and further augmented at 50 and 100 μM. [Green: WGA, SNA, RCA-1, ST6Gal1 and NEU-1, Blue: Hoechst33342 (nuclei)]. Scale bars: 100 μm. b Immunofluorescent staining of sialic acid-related enzyme in HUVECs after GEM exposure. ST6Gal1: In the control state, cytoplasmic localization of ST6Gal1 was detected as a granular dot, corresponding to the Golgi apparatus. The immunofluorescent staining of ST6Gal1 did not significantly change at 5 μM but significantly attenuated in 50 uM and 100 uM of GEM. NEU-1 (sialidase): NEU-1 was weakly present in the cytoplasm in the control state, and the staining did not significantly change after GEM exposure at 5, 50, and 100 μM. Scale bars: 100 μm. c mRNA expression of sialic acid-related enzymes after GEM exposure (RT-PCR). ST6Gal1 mRNA expression was detected in the control state and did not change at 5 uM GEM but decreased at 50 and 100 uM of GEM, whereas NEU-1 (sialidase) mRNA expression was stable and maintained similarly to the control state even after GEM exposure in any concentration
Fig. 5
Fig. 5
Inflammatory cytokine mRNA expression in HUVECs after GEM exposure. IL-6 and IL-1β mRNA expression were barely detectable in the control state; however, after the exposure to GEM, remarkable increases in IL-6 and IL-1β mRNA expression were observed in 5 μM, and similar increases were still detected in both 50 and 100 μM
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Mini E, Nobili S, Caciagli B, Landini I, Mazzei T. Cellular pharmacology of gemcitabine. Ann Oncol. 2006;17(5):v7–v12. doi: 10.1093/annonc/mdj941. - DOI - PubMed
    1. Toschi L, Finocchiaro G, Bartolini S, Gioia V, Cappuzzo F. Role of gemcitabine in cancer therapy. Future Oncol. 2005;1:7–17. doi: 10.1517/14796694.1.1.7. - DOI - PubMed
    1. Fung MC, Storniolo AM, Nguyen B, Arning M, Brookfield W, Vigil J. A review of hemolytic uremic syndrome in patients treated with gemcitabine therapy. Cancer. 1999;85:2023–2032. doi: 10.1002/(SICI)1097-0142(19990501)85:9<2023::AID-CNCR21>3.0.CO;2-2. - DOI - PubMed
    1. Perazella MA. Onco-nephrology: renal toxicities of chemotherapeutic agents. Clin J Am Soc Nephrol. 2012;7:1713–1721. doi: 10.2215/CJN.02780312. - DOI - PubMed
    1. Izzedine H, Isnard-Bagnis C, Launay-Vacher V, Mercadal L, Tostivint I, Rixe O, Brocheriou I, Bourry E, Karie S, Saeb S, Casimir N, Billemont B, Deray G. Gemcitabine-induced thrombotic microangiopathy: a systematic review. Nephrol Dial Transplant. 2006;21:3038–3045. doi: 10.1093/ndt/gfl507. - DOI - PubMed

MeSH terms