Theranostics 2020, Vol. 10, Issue 20 9083 Ivyspring International Publisher Theranostics 2020; 10(20): 9083-9099. doi: 10.7150/thno.46467 Research Paper Lentinan-functionalized Selenium Nanoparticles target Tumor Cell Mitochondria via TLR4/TRAF3/MFN1 pathway Hui-Juan Liu1,2*, Yuan Qin1,2*, Zi-Han Zhao1,2*, Yang Zhang4*, Jia-Huan Yang1,2, Deng-Hui Zhai1,2, Fang Cui1,2, Ce Luo1, Man-Xi Lu1, Piao-Piao Liu1, Heng-Wei Xu1,2, Kun Li1,2, Bo Sun2, Shuang Chen2, Hong-Gang Zhou1, Cheng Yang1,2 and Tao Sun1,2,3 1. State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China. 2. Tianjin Key Laboratory of Early Druggability Evaluation of Innovative Drugs and Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China. 3. Department of Gastroenterology and Hepatology, General Hospital, Tianjin Medical University, Tianjin Institute of Digestive Disease, Tianjin, China. 4. Department of Anesthesiology, Tianjin Fourth Central Hospital, Tianjin, China. *These authors have contributed equally to this work. Corresponding authors: Tao Sun, E-mail: [email protected]; Cheng Yang, E-mail: [email protected]; Hong-gang Zhou, E-mail: [email protected]; Hui-juan Liu, E-mail: [email protected]. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2020.03.29; Accepted: 2020.06.26; Published: 2020.07.11 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. Key words: malignant ascites; ovarian cancer; lentinan; selenium nanoparticles; mitochondria targeting pathway Introduction Ascites caused by malignant tumor is called of vascular permeability [1]. The main causes of malignant ascites. The invasion of tumor tissue into ascites are the alteration of the microvascular peritoneum or abdominal cavity can lead to the networks’ permeability and lymphatic obstruction [2, damage of vascular endothelial cells and the increase 3]. Malignant tumors, such as ovarian and colon http://www.thno.org Theranostics 2020, Vol. 10, Issue 20 9084 cancers, could secrete vasoactive factors that alter the could affect the mitochondrial function, nanoparticles microvascular network permeability and lead to are mainly degraded in the lysosome, which may transudative and exudative ascites [4, 5]. Ovarian induce cell pyroptosis and necroptosis [24, 25]. malignancy is the most common cause (37%) of Inflammatory cytokines caused by pyroptosis and malignant ascites, which leads to poor prognosis and necroptosis may reduce the efficacy of ascites short average survival time [6]. Several methods, such treatment. Therefore, developing a new kind of as intraperitoneal (i.p.) hyperthermic perfusion functionalized SeNPs for malignant ascites treatment chemotherapies, diuresis and abdominal puncture that could selectively enter the mitochondria and drainage, are commonly used for the clinical escape from lysosomes is an important strategy. This treatment of malignant ascites. However, study demonstrated that Selene could efficiently chemotherapy exhibits severe side effects and can’t target the mitochondria via the TLR4/TNF receptor- effectively inhibit inflammatory cytokines; it may associated factor 3 (TRAF3)/mitofusin (MFN1) even promote recurrence and inflammatory cytokine pathway, indicating that TLR4/TRAF3/MFN1 may storms. be a specific nanodrug delivery pathway targeting the Pyroptosis is a kind of necrotic and mitochondria. inflammatory-programmed cell death induced by inflammatory caspases [7]. Pyroptosis is accompanied Methods by a large number of inflammatory cytokines that Materials may stimulate ascites production [8]. Chemo- therapeutic drugs could induce pyroptosis of tumor Mitochondrial membrane potential assay kit cells [9-11]. This phenomenon may explain why (C3601), Fast silver staining kit (P0017S), and cell ascites rebound after chemotherapy. By contrast, mitochondria isolation kit (C2006) were purchased mitochondrion-mediated apoptosis is an immuno- from Beyotime (Shanghai, China). Reactive oxygen logically silent process that may be helpful for species (ROS) assay kit (KGT010-1) and clearing tumor cells and ascites [12]. Thus, inducing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium apoptosis rather than pyroptosis of the cancer cells is bromide (KGA312) were purchased from KeyGen an important approach to inhibit malignant ascites. Biotech (Nanjing, China). Duolink In Situ Probemaker Lentinan (LNT) is a special type of β-glucan (DUO92010/DUO92009) and Duolink flowPLA obtained from Lentinus edode and has a wide variety of detection kit-Red (DUO94001) were purchased from biological and physiological activities [13]. LNT is Sigma–Aldrich (Saint Louis, USA). All siRNA and used in clinics for the treatment of malignant ascites in shRNA were purchased from Origene (Beijing, China, which has high security but low efficiency [14, China). Anti-OPA1 [DF8587, 1:500 for Western blot 15]. Selenium (Se) has a potential application in cancer (WB), 1/100 for immunofluorescence (IF), and 1:200 therapy due to its excellent biological activity and low for immunoprecipitation (IP)], anti-TRAF3 (DF7181, toxicity [16, 17]. Moreover, the biocompatibility and 1:1000 for WB, 1:100 for IF, and 1:200 for IP), anti- efficacy of Se nanoparticles (SeNPs) are better than MFN1 (DF7543, 1:1000 for WB, 1:100 for IF, and 1:200 those of inorganic and organic Se compounds [18-21]. for IP), anti-TLR4 (AF7017, 1:1000 for WB, 1:100 for IF, However, the effect of SeNPs in malignant ascites has and 1:200 for IP) monoclonal antibodies and goat anti- not been reported. LNT could inhibit inflammatory rabbit/mouse-horseradish peroxidase-conjugated cell infiltration and the transformation of secondary antibody (S0001/S0002, 1:5000 dilution) inflammation and cancer by targeting toll-like were purchased from Affinity (Cincinnati, USA). receptor-4 (TLR4), which is highly expressed in tumor Preparation of Selene cells and tumor-infiltrating immune cells [14, 22]. Selene was mainly prepared following the Previous studies have also shown that LNT could procedure described by Jia et al. [23] with some contribute to the stable dispersion of SeNPs in water modification. In brief, freshly prepared LNTs (2 [23]. mg/mL) in aqueous solution (20 mL) were denatured On the basis of the above research, LNT- into single chains at 140 °C. After cooling to room functionalized SeNPs (hereinafter referred to as temperature, the LNT solution was mixed with 500 μL Selene) may exhibit anti-inflammatory and apoptosis- of selenite sodium (0.1 M) and stirred for 5 min. inducting effects, which could contribute to the Approximately 1 mL of ascorbic acid aqueous treatment of malignant ascites. Thus, in this work, the solution (0.2 M) and 20 μL of glutaraldehyde (50%) effect of Selene on malignant ascites was assessed. The solution was added dropwise to the resulting mixture, results showed that Selene could efficiently inhibit the which was then stirred for 24 h at room temperature. ascites in the Ehrlich ascites cancer (EAC) and The reacted product was dialyzed using regenerated OVCAR-3 malignant ascites models. Although SeNPs cellulose tubes (Mw cutoff of 8000) against ultrapure http://www.thno.org Theranostics 2020, Vol. 10, Issue 20 9085 water for 2 days. Then, the resulting solution was transferred to another tube and centrifuged at 11,000× freeze dried using a lyophilizer to obtain SeNPs/LNT g under 4 °C for 10 min, and the precipitate was composites (red powder, Selene). The product was isolated mitochondria. mixed with 6-coumarin to obtain green fluorescent Selene. Immunofluorescence assay OVCAR-3