苏州大学 尹斌 教授
Ferroferric oxide (Fe3O4) nanoparticles induce pro-survival autophagy in human blood cells by positively modulating the Beclin 1/Bcl-2/VPS34 complex（毛新良课题组）
毛新良教授课题组最近发现，Fe3O4纳米颗粒可以通过诱导LC3的脂质化而促发自噬反应。Fe3O4纳米颗粒诱导自噬伴随Beclin 1、VPS34的上调及Bcl-2的下调。进一步的研究表明Fe3O4纳米颗粒可以部分阻止抗癌药物bortezomib 及 doxorubicin引起的细胞凋亡。因此，该研究表明Fe3O4纳米颗粒通过调节Beclin 1/Bcl-2/VPS34轴，在淋巴细胞和髓系细胞引起促进癌细胞生存的自噬反应。这为我们在临床血癌患者中安全应用Fe3O4纳米颗粒提供了参考和依据。
主要参与该工作的有Min Shi, Liang Cheng, Zubin Zhang, Biyin Cao, Zhuang Liu, Xinliang Mao，等。
Abstract: Magnetic iron oxide nanoparticles (MIONPs) are emerging as novel materials with great potentials for various biomedical applications, but their biological activities are largely unknown. In the present study, we found that Fe3O4 nanoparticles (Fe3O4 NPs) induced autophagy in blood cells. Fe3O4 NPs were coated with dopamine (DA), 2,3-dimercaptosuccinnic acid (DMSA), or polyethylene glycol (PEG) and were well dispersed in water, and serum. These NPs triggered autophagy by inducing LC3 ipidation. In consistent with this finding, the protein p62, a monitor of autophagy flux, as degraded by Fe3O4 NPs and this degradation could be abolished by autophagy nhibitors. Mechanistically, Fe3O4 NPs-induced autophagy was accompanied by ncreased Beclin 1 and VPS34, which plays an important role in the formation of ouble-membrane of phagophore, and decreased Bcl-2, which forms a critical complex n autophagy initiation. Further studies demonstrated that Fe3O4 NPs partially blocked ell death induced by anti-cancer drugs bortezomib and doxorubicin, both of which are idely used for patients with multiple myeloma. Therefore, this study demonstrated hat Fe3O4 NPs can induce pro-survival autophagy in both lymphocytes and myelocytes y modulating the Beclin l/Bcl-2/VPS34 complex. This study suggests that a caution hould be taken when Fe3O4 NPs are used in blood cancer patients.
Results and Figures:
1. Characterization of surface-modified Fe3O4 NPs
Synthesized Fe3O4 NPs were first analyzed with TEM for the size and morphology. As shown in Figures 1A, B and C, Fe3O4 NPs coated with various modifications were uniformly dispersed, the average sizes of the particles are <10 nm. Further studies by dynamic light scattering revealed the hydrodynamic sizes varied depending on the modifications. The average DLS sizes of DA and DMSA modified-nanoparticles were 25±4 and 26±4 nm, respectively, while the PEG modification increased the average DLS size to 50±7 nm (Fig 1D). Zeta potentials of each type of Fe3O4 NPs in water were shown in Fig 1E. As expected, the modification affected the surface charge. Compared with the Fe3O4-DA and Fe3O4-DMSA-DMSA particles, the Fe3O4-DA-PAA-PEG particles carried a relative low potential. Further studies showed that these modified Fe3O4 NPs could dissolve in different types of solutions including water, phosphate buffered saline (PBS), RPMI-1640 cell medium, and fetal bovine serum (FBS), all solutions exhibited excellent stability in all tested physiological solutions (Fig 1F-1H) within 24 hours.
2. Fe3O4 NPs induce autophagy in blood cancer cells
Nanoparticles are not toxic to many cell types, but recent investigations showed that they can induce apoptosis and/or autophagy in certain cells. To find out whether the Fe3O4 NPs induced autophagy in blood cancer cells, leukemia (OCI-AML2 and K562) and multiple myeloma (MM) (OPM2, 8226, JJN3) cell lines were treated with 100 μg/mL of Fe3O4 NPs with specific modifications including DA, DMSA, or PEG for 9 hours. To view autophagy induced by Fe3O4 NPs, the expression level of LC3-II, a phosphatidylethanolamine (PE)-conjugated form from microtubule-associated protein 1A/1B-light chain 3 (LC3) was measured because it is a common indicator of autophagy. Immunoblotting suggested that LC3-II was induced in all cells treated with Fe3O4 NPs (Figure 2A). This induction of LC3-II was in a time- and concentration-dependent manner (Figures 2B and C). LC3-II could be induced by Fe3O4 NPs at a concentration as low as 12.5 g/mL within 9 hours (Figure 2B), or within 3 hours at the 100 g/mL of all three types of particles (Figure 2C). To further visualize whether increased LC3-II was attached to the autophagosome membrane, OPM2 cells were starved or treated with Fe3O4 NPs at 100 μg/mL for 9 hours, followed by immunofluorescence staining. As shown in Figure 3, starvation, as predicted, led to increased LC3-II puncta appearing as green dots in the confocal microscope. Similar to starvation, Fe3O4 NPs at all modifications led to increased LC3-II puncta, a critical marker of autophagy. Therefore, Fe3O4 NPs probably induce autophagy in blood cancer cells.
3. Fe3O4 NPs-induced autophagy is blocked by inhibitors of autophagy or lysosomes
It is well known that the autophagy can be blocked by specific autophagy inhibitors or lysosome inhibitors. To further confirm Fe3O4 NPs-induced autophagy, myeloma cell 5 line OPM2 was treated with Fe3O4 NPs alone or along with 3-methyladenine (3-MA), an inhibitor of autophagy, or bafilomycin A1 (BafA), a lysosome inhibitor. As shown in Figure 4, Fe3O4 NPs induced LC3-II and decreased p62, a protein degraded via the autophagy pathway and an important biomarker of autophagy. When 3-MA was added, the changes of LC3-II and p62 were abolished (Figures 4A, B and C). While BafA increased the p62, suggesting p62 degradation was blocked by the lysosome inhibitor. These changes along with the results in Figures 2 and 3 demonstrated that Fe3O4 NPs induced autophagy in blood cancer cells.
4. Fe3O4 NPs target the Beclin 1/VPS34/Atg14 complex
Autophagy is mediated by a series of important cell signaling pathway. Its initiation is triggered by nutrient depletion via the mTOR signaling, while the elongation of autophagosomes is mainly modulated by the Beclin 1/VPS34/Atg14 complex . To find out the mechanisms involved in Fe3O4 NPs-induced autophagy, we first evaluated the effects of the AKT/mTOR signaling after Fe3O4 NPs treatment for 9 hours in which time Fe3O4 induced autophagy as shown in Figures 2 and 3. However, immunoblotting revealed that mTOR activation was not affected by Fe3O4 NPs at 9 hours after treatment (Figure 5A). Because 3-MA, an inhibitor of VPS34, blocked autophagy induced by Fe3O4 NPs, it suggested that the VPS34 complex might play a key role in this autophagy. VPS34 acts by forming a complex with Beclin 1, Atg14 and other proteins to promote elongation of autophagosome , therefore, we next evaluated the expression levels of the components in the VPS34 complex. Immunoblotting revealed that Beclin 1, VPS34, and Atg14 were induced while the Bcl-2, a negative modulator of Beclin 1, was decreased by these NPs (Figure 5B). And these changes were time- and concentration- dependent (Figures 5C and D). We next checked the protein levels of Atg14 and Beclin 1 after cells were treated for 3 to 24 hours and found that both Beclin 1 and Atg14 maintained a high level throughout this period, which was consistent with LC3-II expression. These results thus suggested the VPS34/Beclin 1 complex was critical for Fe3O4 NP-induced autophagy.
5. Fe3O4 NPs prevent cell apoptosis of myeloma cells induced therapeutic drugs
Autophagy has been proposed to deliver a survival or a death signal depending on the circumstance of cells . To evaluate the effects of autophagy on cell apoptosis, we first measured cell proliferation in the presence of these chemically modified Fe3O4 NPs. It showed that these Fe3O4 NPs had no effects on cell proliferation even at a concentration up to 2 g/mL (Data not shown). PARP and caspase-3, both of which are common biomarkers of cell apoptosis, were not cleaved by Fe3O4 NPs at 100 μg/mL for 9 hours (Figure 6A). When an anti-myeloma drug bortezomib, dexamethasone or doxorubicin was added, both PARP and caspase-3 was cleaved, suggesting that cells underwent apoptosis. However, this apoptosis could be partly attenuated by these nanoparticles because the cleaved forms of PARP and Caspase-3 were decreased (Figures 6B, C and D). Therefore, this experiment indicated that Fe3O4 NP-induced autophagy exhibited an anti-apoptotic or a pro-survival function at least in blood cancer cells.
6. Bare Fe3O4 NPs induces pro-survival autophagy
Above studies demonstrated that these Fe3O4 NPs with various chemical modifications were able to induce autophagy in blood cancer cells. Because these modifications included negatively charged (DMSA), and positively charged (PEG and DA), which suggested that modification might not be important for the autophagy induced by these Fe3O4 NPs. To find out whether this autophagy was triggered by these core Fe3O4 NPs or by the modifications, we next examined the effects of bare Fe3O4 NPs on autophagy in the same cell line – OPM2. As shown in Figures 7A and B, bare Fe3O4 NPs induced the expression of both LC3-II and Beclin 1 in a concentration- and time-course dependent manner. Similar to the chemically modified Fe3O4 NPs, bare Fe3O4 NPs also led to degradation of p62, which could be abrogated by the autophagy inhibitor 3-MA or lysosome inhibitor BafA (Figure 7C). Notably, these bare Fe3O4 NPs also displayed anti-apoptotic activity (Figure 7D). Therefore, these studies showed that Fe3O4 NPs induced autophagy independent of the surface modifications.
7. Fe3O4 NPs induce autophagy in primary blood cells
Lastly, we questioned whether these Fe3O4 NPs induce autophagy in healthy blood cells. To this end, we isolated myeloid and lymphoid cells from mice and incubated these cells with Fe3O4 NPs for 9 hours. Similar to the effects of Fe3O4 NPs on blood cancer cells, all these Fe3O4 NPs led to increased expression of LC3-II and Beclin 1, and downregulated the expression of Bcl-2 in the primary healthy blood cells (Figure 8). These results indicated that Fe3O4 NPs also induced autophagy in primary healthy blood cells.