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The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

Shang C, Chen Q, Dell A, Haslam SM, De Vos WH, Van Damme EJ - PLoS ONE (2015)

Bottom Line: In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins.Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula.Our data suggest that one of these pathways involves the induction of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ABSTRACT
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

No MeSH data available.


Related in: MedlinePlus

Dose response curve of the effect of S. nigra proteins on HeLa and NHDF cell viability after 24 and 48 h.(A) Dose-response curves for HeLa cells incubated for 24 and 48h with different concentrations of S. nigra RIPs/lectins. (B) Log concentration – cell viability curve of NHDF cells incubated for 24 and 48 h with different concentrations of S. nigra RIPs/lectins. % ctrl (treated/control X 100) = ratio of surviving treated cells/ surviving cells percent in control. All data are expressed as means ± SE of 3 biological replicates in 4 technical replicates (n = 12). (C) Transmission light microscopy images of HeLa cells grown in the absence (control) and presence of 1.5 μM SNA-V for 24 h. Scale bars represent 100 μm.
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pone.0132389.g002: Dose response curve of the effect of S. nigra proteins on HeLa and NHDF cell viability after 24 and 48 h.(A) Dose-response curves for HeLa cells incubated for 24 and 48h with different concentrations of S. nigra RIPs/lectins. (B) Log concentration – cell viability curve of NHDF cells incubated for 24 and 48 h with different concentrations of S. nigra RIPs/lectins. % ctrl (treated/control X 100) = ratio of surviving treated cells/ surviving cells percent in control. All data are expressed as means ± SE of 3 biological replicates in 4 technical replicates (n = 12). (C) Transmission light microscopy images of HeLa cells grown in the absence (control) and presence of 1.5 μM SNA-V for 24 h. Scale bars represent 100 μm.

Mentions: To assess the antiproliferative activity of the S. nigra RIPs (SNA-I, SNA-V and SNLRP) and lectins (SNA-II and SNA-IV) on HeLa and NHDF cells, spectrophotometric viability assays were performed after incubation with different concentrations (0.1–2 μM) of the proteins (Fig 2A and 2B). In HeLa cells, all proteins, except for SNA-IV, induced significant (p<0.05) cytotoxicity after 48 h incubation at the lowest protein concentration tested (0.1 μM). Furthermore, SNA-IV also became cytotoxic at concentrations > 1.5 μM. There was a clear dose- and time-dependent effect on HeLa cell viability. The degree of cytotoxicity after 48 h was as follows: SNA-V > SNA-II > SNA-I > SNLRP > SNA-IV (Table 1). The cytotoxic effect on HeLa cells was accompanied by clear morphological changes such as cell rounding and blebbing (Fig 2C). As evidenced by the increased LC50 values (Table 1), NHDF cells were much less susceptible to S. nigra proteins than HeLa cells. There was no statistically significant effect of SNA-IV and SNLRP on NHDF cell viability and proliferation after 24 h whereas all the other S. nigra proteins caused a significant cytotoxicity, though at much higher protein concentrations compared to HeLa cells. Only after 48 h, a significant effect on cell viability was witnessed for SNA-V (p<0.001) at the lowest protein concentration (0.05 μM).


The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

Shang C, Chen Q, Dell A, Haslam SM, De Vos WH, Van Damme EJ - PLoS ONE (2015)

Dose response curve of the effect of S. nigra proteins on HeLa and NHDF cell viability after 24 and 48 h.(A) Dose-response curves for HeLa cells incubated for 24 and 48h with different concentrations of S. nigra RIPs/lectins. (B) Log concentration – cell viability curve of NHDF cells incubated for 24 and 48 h with different concentrations of S. nigra RIPs/lectins. % ctrl (treated/control X 100) = ratio of surviving treated cells/ surviving cells percent in control. All data are expressed as means ± SE of 3 biological replicates in 4 technical replicates (n = 12). (C) Transmission light microscopy images of HeLa cells grown in the absence (control) and presence of 1.5 μM SNA-V for 24 h. Scale bars represent 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493096&req=5

pone.0132389.g002: Dose response curve of the effect of S. nigra proteins on HeLa and NHDF cell viability after 24 and 48 h.(A) Dose-response curves for HeLa cells incubated for 24 and 48h with different concentrations of S. nigra RIPs/lectins. (B) Log concentration – cell viability curve of NHDF cells incubated for 24 and 48 h with different concentrations of S. nigra RIPs/lectins. % ctrl (treated/control X 100) = ratio of surviving treated cells/ surviving cells percent in control. All data are expressed as means ± SE of 3 biological replicates in 4 technical replicates (n = 12). (C) Transmission light microscopy images of HeLa cells grown in the absence (control) and presence of 1.5 μM SNA-V for 24 h. Scale bars represent 100 μm.
Mentions: To assess the antiproliferative activity of the S. nigra RIPs (SNA-I, SNA-V and SNLRP) and lectins (SNA-II and SNA-IV) on HeLa and NHDF cells, spectrophotometric viability assays were performed after incubation with different concentrations (0.1–2 μM) of the proteins (Fig 2A and 2B). In HeLa cells, all proteins, except for SNA-IV, induced significant (p<0.05) cytotoxicity after 48 h incubation at the lowest protein concentration tested (0.1 μM). Furthermore, SNA-IV also became cytotoxic at concentrations > 1.5 μM. There was a clear dose- and time-dependent effect on HeLa cell viability. The degree of cytotoxicity after 48 h was as follows: SNA-V > SNA-II > SNA-I > SNLRP > SNA-IV (Table 1). The cytotoxic effect on HeLa cells was accompanied by clear morphological changes such as cell rounding and blebbing (Fig 2C). As evidenced by the increased LC50 values (Table 1), NHDF cells were much less susceptible to S. nigra proteins than HeLa cells. There was no statistically significant effect of SNA-IV and SNLRP on NHDF cell viability and proliferation after 24 h whereas all the other S. nigra proteins caused a significant cytotoxicity, though at much higher protein concentrations compared to HeLa cells. Only after 48 h, a significant effect on cell viability was witnessed for SNA-V (p<0.001) at the lowest protein concentration (0.05 μM).

Bottom Line: In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins.Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula.Our data suggest that one of these pathways involves the induction of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ABSTRACT
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

No MeSH data available.


Related in: MedlinePlus