Lentinan-functionalized Selenium Nanoparticles target Tumor Cell Mitochondria via TLR4/TRAF3/MFN1 pathway

Abstract

Rationale: Malignant ascites caused by cancer cells results in poor prognosis and short average survival time. No effective treatment is currently available for malignant ascites. In this study, the effects of lentinan (LNT)-functionalized selenium nanoparticles (Selene) on malignant ascites were evaluated. Furthermore, the mechanism of Selene targeting mitochondria of tumor cells were also investigated. Methods: Selene were synthesized and characterized by TEM, AFM and particle size analysis. The OVCAR-3 and EAC cells induced ascites models were used to evaluate the effects of Selene on malignant ascites. Proteomic analysis, immunofluorescence, TEM and ICP-MS were used to determine the location of Selene in tumor cells. Mitochondrial membrane potential, ROS, ATP content, and caspase-1/3 activity were detected to evaluate the effect of Selene on mitochondrial function and cell apoptosis. Immunofluorescence, Co-IP, pull-down, duolink, Western blot, and FPLC were used to investigate the pathway of Selene targeting mitochondria. Results: Selene could effectively inhibit ascites induced by OVCAR-3 and EAC cells. Selene was mainly located in the mitochondria of tumor cells and induced apoptosis of tumor cells. The LNT in Selene was involved in caveolae-mediated endocytosis through the interaction between toll-like receptor-4 (TLR4) and caveolin 1 (CAV1). Furthermore, the Selene in the endocytic vesicles could enter the mitochondria via the mitochondrial membrane fusion pathway, which was mediated by TLR4/TNF receptor associated factor 3 (TRAF3)/mitofusin-1 (MFN1) protein complex. Conclusion: Selene is a candidate anticancer drug for the treatment of malignant ascites. And TLR4/TRAF3/MFN1 may be a specific nano-drug delivery pathway that could target the mitochondria.

Keywords: lentinan; malignant ascites; mitochondria targeting pathway; ovarian cancer; selenium nanoparticles.

Figure 1

Preparation and characterization of Selene. A. Synthetic process of lentinan-functionalized Se nanoparticles (Selene). B. TEM images of Selene. C. AFM images of Selene. D. Size distribution of Selene. E. EDX detection results of Selene. F. Stability detection results of Selene in saline (0.9% NaCl) at room temperature. G. FTIR spectra of Selene. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 2

Selene inhibited ascites induced by EAC in vivo. A. Schematic diagram of model establishment and drug administration. The mice were treated with Selene, lentinan, or SeNPs from the fourth day after the modeling, when ascites began to appear. B. Representative images of mice in the different groups. C-E. Body weight, volume of ascites, and cancer cell numbers in ascites of each group. F. Schematic diagram of model establishment and drug administration. The mice were treated with Selene from the seventh day after the modeling, when a large amount of ascites appeared. G. Representative images of mice in different groups. H-J. Changes in body weight, volume of ascites, and cancer cell number in ascites of different groups. K. Schematic diagram of model establishment and drug administration. The mice were treated with Selene after the ascites were removed from the seventh day after the modeling. L. Representative images of mice in the different groups. M-O. Changes in body weight, volume of ascites, and cancer cell numbers in ascites of the different groups. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 3

Selene inhibited ascites induced by ovarian cancer cells in vivo. A and B. Results of B ultrasound and abdominal volume of ascites induced by ovarian cancer cells (OVCAR-3) in different groups. C. Representative images of mice in different groups. D and E. Volume of ascites and cancer cell numbers in ascites. F. Results of cell apoptosis detection of the cancer cells in ascites. G. The photographs of Evans blue dye leakage in the intestines and abdominal walls. H. The photographs of the aspirated ascites before (upper panel) and after (lower panel) centrifugation. I. Comparison of Evans blue amounts in ascites of each group. J. The photographs of the FITC-dextran intensity in 20 mL of ascites of each sample. K. Analysis results of cytokine antibody chip assay of Selene-treated group compared with the model group. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 4

Selene could influence mitochondrial function and induce apoptosis of OVCAR-3 cells. A and B. Proteomic analysis results of cells treated with Selene compared with the control group. Differentially expressed proteins were analyzed using GO and KEGG databases to explore the main functions and signaling pathways affected by Selene. C and D. Detection results of mitochondrial membrane potential and ROS in Selene treated cells. E. Uptake efficiency of Selene and SeNPs in OVCAR-3 cells. F. Relative Se content in the mitochondria and lysosomes of OVCAR-3 cells. G. Ultrathin sectioning of cells detected by TEM. H. Colocation results of Selene/SeNPs and lysosomes. I. Colocation results of Selene/SeNPs and mitochondria. J. SEM images of OVCAR-3 cells. K. Expression of caspase 1 and 3 detected by Western blot. L. Results of ATP level in cells treated with Selene and SeNPs. M and N. Caspase 1 and caspase 3 activities in cells treated with Selene and SeNPs. O. The content of IL-1β in each group. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 5

Selene targeting the mitochondria via the caveolae-mediated endocytosis pathway. A. Immunofluorescence results of Selene accumulation in cells at different time points. B and C. Se content in the whole cell and mitochondria detected using ICP-MS. D. Results of ultrathin sectioning of cells treated with Selene detected by the TEM. E. Se content in cells after low-temperature (4 °C) and energy-depletion agent (NaN₃ + deoxyglucose) treatment. F. Se content in cells treated with clathrin-mediated endocytic inhibitors (sucrose and chlorpromazine), caveolae-mediated endocytic inhibitors (MβCD and nystatin), and macropinocytosis inhibitor (EIPA and amiloride). G. Co-IP results of TLR4 and CAV1 detected by Western blot. H and I. Immunofluorescence double-labeling results of TLR4 and CAV1. J and K. Duolink PLA results of TLR4 and CAV1 (Selene-L 10 µM, Selene-H 15 µM). L and M. Colocation results of Selene and the mitochondria after SiTLR4 treatment, as detected via immunofluorescence. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 6

Selene was specifically enriched in the mitochondria through TLR4/TRAF3/MFN1 protein complex-mediated membrane fusion. A. Results of potential bridge-linked protein analysis from FpClass database. B. Pull-down assay results (IP by the anti-Flag beads) of the input, control vector, and Flag-TLR4 overexpression groups detected via silver staining to identify proteins interacting with TLR4. C-E. Duolink PLA results of TLR4/TRAF3 and TRAF3/MFN1 after Selene treatment (Selene-L 10 µM, Selene-H 15 µM). F. Whole-cell lysates from OVCAR-3 were immunoprecipitated by anti-TRAF3, anti-TLR4, anti-MFN1, and anti-OPA1 antibodies, followed by Western blot with antibodies against the indicated proteins to verify the interactions of proteins. G. Cell lysate was separated by FPLC. Then, TRAF3, TLR4, and MFN1 in the fractions were detected using Western blot. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 7

Selene was specifically enriched in the mitochondria via TLR4/TRAF3/MFN1 pathway. A. Colocalization of MFN1/TRAF3 and TLR4/TRAF3 was analyzed by immunostaining of OVCAR-3 cells and detected using confocal microscopy. B. Colocation of Selene and mitochondria in OVCAR-3 cells after TRAF3 and MFN1 knockdown. C and D. Se content in the whole cells and mitochondria as detected using ICP-MS in TRAF3 and MFN1 knocked down OVCAR-3 cells. E and F. The effect of Selene on OVCAR-3 ascites model when TLR4 and TRAF3 in OVCAR-3 cells were knocked down. G. Results of cell apoptosis detection in the OVCAR-3 ascites model when TLR4 and TRAF3 in OVCAR-3 cells were knocked down. Results are shown as means ± SD (*P < 0.05, **P < 0.01).

Figure 8

Model of Selene targeting the mitochondria via TLR4/TRAF3/MFN1 pathway.