High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells

Abstract

BackgroundPseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated.MethodsCell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated.ResultsThe strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth.ConclusionPFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2099-2) contains supplementary material, which is available to authorized users.

Targeting of crosstalk between tumor and tumor microenvironment by β‐D mannuronic acid (M2000) in murine breast cancer model

“…For instance, recent observations indicate that blocking of mannose ligand residues on the endothelial cell surface by metaperiodate treatment significantly reduced tumor cell adhesion and metastasis [46]. Moreover, adherence of tumor cells to extracellular matrix was dramatically abolished by soluble mannose-binding lectin treatment [47]. It has been supported by antitumorigenic effects of mannose-recognizing plant lectins [48], use of mannosylated chitosan for prostate cancer immunotherapy [49], application of mannosylated radiotracers for solid tumor staging [50], and positive correlation between levels of high mannosylation glycoproteins and the tumor burden in both mouse and human during breast cancer progression [51].…”

Anti-influenza virus activity of high-mannose binding lectins derived from genus Pseudomonas

“…In fact, the treatment with PFL disturbed various cellular responses such as proliferation or autophagy. The change of expression in many genes was detected to a varying degree (Sato et al, 2016). However, the GRFT treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines.…”

Copolymers enhance selective bacterial community colonization for potential root zone applications

“…Importantly, freely absorbed mannans increase the translocation and colonization of these bacterial species greater than grafted chains as shown by their lower surface density but higher internal population. The behaviour of P. fluorescens is consistent with the presence of high mannose binding adhesins which promote localization 32 , 33 , whereas these adhesins have been shown to be absent in the genome sequence of B. subtilis 34 . The relative internal cell population in hydrogel region between 50 to 170 µm, i.e excluding the interfacial area of 0–49 µm, were 1,780, 12,385, and 65,965 cells in PAA, PAA-mann graft and PAA-mann free respectively.…”