LRP1-Mediated Endocytosis May Be the Main Reason for the Difference in Cytotoxicity of Curcin and Curcin C on U2OS Osteosarcoma Cells

Abstract

Curcin and Curcin C, both of the ribosome-inactivating proteins of Jatropha curcas, have apparent inhibitory effects on the proliferation of osteosarcoma cell line U20S. However, the inhibitory effect of the latter is 13-fold higher than that of Curcin. The mechanism responsible for the difference has not been studied. This work aimed to understand and verify whether there are differences in entry efficiency and pathway between them using specific endocytosis inhibitors, gene silencing, and labeling techniques such as fluorescein isothiocyanate (FITC) labeling. The study found that the internalization efficiency of Curcin C was twice that of Curcin for U2OS cells. More than one entering pathway was adopted by both of them. Curcin C can enter U2OS cells through clathrin-dependent endocytosis and macropinocytosis, but clathrin-dependent endocytosis was not an option for Curcin. The low-density lipoprotein receptor-related protein 1 (LRP1) was found to mediate clathrin-dependent endocytosis of Curcin C. After LRP1 silencing, there was no significant difference in the 50% inhibitory concentration (IC₅₀) and endocytosis efficiency between Curcin and Curcin C on U2OS cells. These results indicate that LRP1-mediated endocytosis is specific to Curcin C, thus leading to higher U2OS endocytosis efficiency and cytotoxicity than Curcin.

Keywords: Curcin; Curcin C; endocytosis; osteosarcoma U20S cells; ribosome-inactivating proteins.

Figure 1

Cytotoxicity and intracellular fluorescence intensity of FITC-Curcin (FITC-C), FITC-Curcin C (FITC-CC), His-eGFP-Curcin (eGFP-C), and His-eGFP-Curcin C (eGFP-CC). (A) Viability of U2OS cells after 48 h FITC-C and FITC-CC treatment. (B) Viability of U2OS cells after 48 h eGFP-C and eGFP-CC treatment. (C) Flow cytometry histograms of intracellular fluorescence intensity after 1, 3, and 5 h incubation with 0.5 μM eGFP-C and eGFP-CC. (D) Flow cytometry histograms of intracellular fluorescence intensity after 4 h incubation with 0.25 μM, 0.5 μM, and 1 μM eGFP-C and eGFP-CC. All data are the mean ± SD of three independent experiments.

Figure 2

FITC-C and FITC-CC entered U2OS cells in a time- and concentration-dependent manner. (A) Observation of FITC-C-, FITC-CC-, and FITC-labeled bovine serum albumin (FITC-BSA) in U2OS cells under a fluorescence microscope. The nucleus was stained with DNA-specific fluorescent dye Hoechst 33258. Scale bar, 75 μm. (B) Flow cytometry histograms of 0.5 μM FITC-Curcin and FITC-Curcin C fluorescence intensity in U2OS cells after 3 h, 12 h, and 24 h incubation. (C) Mean fluorescence intensity (MFI) of 0.5 μM FITC-Curcin and FITC-Curcin C in U2OS cells after 3 h, 12 h, and 24 h incubation. (D) Flow cytometry histograms of FITC-Curcin and FITC-Curcin C fluorescence intensity in U2OS cells after 12 h incubation with various concentrations (0.125 μM, 0.25 μM, 0.5 μM, and 1 μM). (E) The MFI of FITC-Curcin and FITC-Curcin C in U2OS cells after 12 h incubation with various concentrations (0.125 μM, 0.25 μM, 0.5 μM, and 1 μM).

Figure 3

Effects of different endocytosis inhibitors on the Curcin and Curcin C cytotoxicity against U2OS cells. Control check (CK); chloroquine (CQ); chlorpromazine (CPZ); methyl-β-cyclodextrin (MβCD). (A) Proliferation inhibition rate of different endocytosis inhibitor-pretreated U2OS cells after 48 h Curcin treatment. (B) IC₅₀ of Curcin after 48 h Curcin treatment on different endocytosis inhibitor-pretreated U2OS cells. (C) Proliferation inhibition rate of different endocytosis inhibitor-pretreated U2OS cells after 48 h Curcin C treatment. (D) IC₅₀ of Curcin C after 48 h Curcin C treatment on different endocytosis inhibitor-pretreated U2OS cells. All data are presented as the mean ± SD of three independent experiments (***, p < 0.001; **, p < 0.01; * p < 0.05).

Figure 4

Expression of LDL family and LRP1 in tumor tissues and the relationship between LRP1 and cytotoxicity of Curcin C. (A) Heat map of LDL receptor family expression in 30 types of tumors and adjacent normal tissues, T—tumor tissues; N—normal tissues. (B) Expression of LRP1 in 35 tumor types and their adjacent normal tissues. The red arrow indicates the expression of LRP1 in sarcoma. (***, p < 0.001; **, p < 0.01; * p < 0.05). (C) Expression of LRP1 in 6 cell lines and IC₅₀ of Curcin C for their corresponding cell lines. These 6 cell lines belong to 4 tumors (SARC: U2OS; LUAD: A549, H1975; COAD: HCT116, HT-29; LAML: MV4-11). IC₅₀ data from Zhang et al., 2017 and Wang et al., 2022 [13,30]; the average of three independent experiments.

Figure 5

LRP1 is one of the Curcin C endocytic receptors. (A) Western Blot analysis of LRP1 expression levels at different time points after siRNA silencing. (B) Proliferation inhibition rate of siRNA-treated cells vs. untreated cells after 48 h treatment with different concentrations of Curcin and Curcin C. The “siRNA-C” and “siRNA-CC” indicate that cells were pretransfected with LRP1-siRNA for 48 h and then treated with C/CC for 48 h, and “NC-C” and “NC-CC” represent cells as the negative control, which are pre transfected with NC-siRNA for 48 h, and then treated with C/CC for 48 h. (C) IC₅₀ of Curcin and Curcin C after 48 h of Curcin and Curcin C treatment in U2OS cells with different concentrations. (D) Flow cytometry was used to detect the fluorescence intensity of FITC-Curcin and FITC-Curcin C after LRP1 silencing in U2OS cells. Cells not treated with Curcin and Curcin C protein as a blank group. (E) The MFI of FITC-Curcin and FITC-Curcin C after LRP1 silencing vs. NC-C and NC-CC. All data are the mean ± SD of three independent experiments (***, p < 0.001). Different letters (a–c) indicate significant differences (p < 0.05; one-way ANOVA with Tukey’s honestly significant difference test).