苏州大学 尹斌 教授
1 Poly-(allylamine hydrochloride) but not poly(acrylic acid)-coated upconversion nanoparticles induce autophagy and apoptosis in human blood cancer cells（毛新良课题组）
基于其在医学领域的各种应用如活细胞成像和光动力学疗法，上转换纳米颗粒UCNPs逐渐得到了关注。但是，其抗癌的直接效果报道较少。本研究合成了NaGdF4:Yb,Er UCNPs，包被了两种材料聚丙烯胺盐酸盐PAH或者聚丙烯酸PAA，并且研究了其在骨髓瘤和白血病细胞中的作用。当人类骨髓瘤细胞和白血病细胞与两种UNCPs孵育的时候，PAH-UCNPs而非PAA-UCNPs促进了自噬标志物LC3-II 的表达，这种升高的表达可被自噬抑制剂3-MA阻断。PAH-UCNPs 也抑制了自噬的关键步骤即AKT和mTOR 的激活。进一步研究表明PAH-UCNPs也降低了Bcl-2、促进了Beclin1和 Atg14 的表达，表明PAH-UCNPs诱导的自噬和升高的PI3KC3/Beclin1活性相关。此外，PAH-UCNPs 诱导了骨髓瘤细胞的凋亡，并且增强了主要的抗癌药物硼替佐米和阿霉素诱导的凋亡。因此，该研究表明PAH-UCNPs 自身可以通过调节AKT/mTOR以及PI3KC3/Beclin1/Atg14信号通路，诱导人血液肿瘤细胞的自噬和凋亡。本研究提示了PAH-UCNPs 在血液肿瘤细胞杀伤中的潜在应用。主要参与该工作的有Min Shi，Liang Cheng，Jiaxiang Juan，Zhuang Liu，Xinliang Mao，该工作具体内容介绍如下：
Upconversion nanoparticles (UCNPs) have gained increased attention due to its various medical applications such as living cell imaging and photodynamic therapy. But little is known about its direct anti-cancer activity. In the present study, we synthesized NaGdF4:Yb,Er UCNPs coating with poly-(allylamine hydrochloride) (PAH) or poly(acrylic acid) (PAA) and investigated their effects in human myeloma and leukemia cells. When human myeloma and leukemia cells were incubated with both types of UCNPs, we found that PAH- but not PAA-UCNPs increased the expression of LC3-II, a hallmark of autophagy. This effect was confirmed by the accumulation of LC3 puncta analyzed by immunofluorescence microscopy. Induction of LC3-II could be blocked by 3-methyl adenosine (3-MA), an autophagy inhibitor. Consistent with this observation, PAH-UCNPs also inhibited both AKT and mTOR activation, the key step in autophagy activation. Further studies demonstrated that PAH-UCNPs also decreased Bcl-2 but increased Beclin1 and Atg14 expression, further suggesting PAH-UCNPs-induced autophagy was associated with increased activity of the PI3KC3/Beclin1 activity. Moreover, PAH-UCNPs induced apoptosis in myeloma cells and enhanced apoptosis induced by bortezomib and doxorubicin, two major anti-myeloma drugs. Therefore, our study suggested that PAH-UCNPs alone can induce both apoptosis and autophagy in human blood cancer cells by modulating the AKT/mTOR and the PI3KC3/Beclin1/Atg14 signaling pathways. This study implies the potential application of PAH-UCNPs in blood cancer-cell killing.
Results and Figures:
(1) PAH- but not PAA-modified UCNPs induce autophagy in human blood cancer cells.
UCNPs display great biocompatibility and limited toxicity was reported. As a major response to many chemical, physical, biological stresses, autophagy has been extensively studies in exposure to nanomaterials. But the effect of UCNPs on authophagy is unclear. To this end, we examined whether UCNPs could induce autophagy in blood cancer cells because blood is the first system exposure to these nanomaterials in their biomedical applications. Multiple myeloma (MM) (OPM2, JJN3) and leukemia (K562) cell lines were incubated for 9 hours at a concentration of 100 μg/mL of PAH- and PAA-UCNPs, respectively. We next examined the protein level of LC3-II, a hallmark of autophagy converted from LC3-I. As shown in Figure 1A, PAH-UCNPs increased the LC3-II levels in all cells tested but it was not observed in cells exposed to PAA-UCNPs. Notably, PAH-UCNPs-raised LC3-II level was time- and concentration-dependent (Fig. 2B). OPM2 cells were exposed to increased concentrations of PAH-UCNPs for 9 hours or 100 μg/mL of PAH-UCNPs for various periods. Immunoblotting revealed that significant LC3-II was observed at a concentration as low as 12.5 μg/mL or in 3 hours at the concentration of 100 μg/mL (Fig. 1B). When autophagy is triggered, LC3-II accumulates and form puncta around nucleus. To verify LC3-II expression, OPM2 cells exposed to PAH- or PAA-UCNPs were subjected to immunofluorescent analysis. As expected, starvation resulted in markedly accumulated LC3-II puncta (Fig. 1C). Similar to the treatment of starvation, PAH-UCNPs also led to significant increase of LC3-II dots. However, PAA-UCNPs failed to increase LC3-II puncta in the same treatment (Fig. 1C). All of the above results thus suggested that PAH- but not PAA-modified UCNPs induced autophagy in blood cancer cells.
(2) PAH-UCNPs require the PI3KC3/Beclin1/Atg14 complex in autophagy
Autophagy is a multiple-step process involved in several key complexes and signaling transduction. For example, Class III of phosphatidylinositol-3 kinase (PI3KC3) forms a complex with Beclin1/Atg14 to mediate autophagasome formation and elongation, thus it is essential for autophagy. To further understand UCNPs-induced autophagy occurrence, OPM2 cells were pre-treated with 3-MA, a well known inhibitor of autophagy by targeting PI3KC3, followed by PAH-UCNPs. As shown in Fig.2A, PAH-UCNPs increased LC3-II level, but it was markedly reduced by 3-MA, further suggested that UCNPs-induced autophagy and it was associated with PI3KC3 signaling. Notably, bafilomycin A, an inhibitor of lysosomes, increased LC3-II expression, suggested UCNPs-induced autophagosome is processed in the lysosomes (Fig.2A). These changes demonstrated that PAH-UCNPs could induce autophagy via PI3KC3 in human blood cancer cells and this type of autophagy could be blocked by inhibitors of autophagy or lysosomes.
The above study suggested PI3KC3 was required for UCNPs-induced autophagy. Because PI3KC3 acts in a complex with Atg6/Beclin1 and Atg14, we questioned whether UCNPs interfered with other components of this complex. OPM2 cells were treated with PAH-UCNPs for 9 hours at a concentration of 100 μg/mL and then evaluated the expression of associated proteins. As shown in Figure 2B, PAH-UCNPs but not PAA-UCNPs induced Beclin1 and decreased Bcl-2 expression (Fig. 2B). PAH-UCNPs induced Beclin1 in a time- and concentration-dependent manner (Fig.2C). In addition to Beclin1, PI3KC3 complex also requires Atg14 in autophagosome formation, therefore, we next measured the effects of PAH-UCNPs on Atg14. It turned out that PAH-UCNPs but not PAA-UCNPs induced the expression of Atg14. All these results above thus suggested PAH-UCNPs induced autophagy by targeting the PI3KC3/Beclin1/Atg14 complex via the induction of Becin1 and Atg14 expression.
(3) PAH-UCNPs induce autophagy by targeting Akt/mTOR signaling
The mTOR kinase has been believed to be the key in autophagy initiation, especially in starvation situations. To find out whether PAH-UCNPs also affect mTOR signaling, OPM2 cells were treated with PAH-UCNPs or PAA-UCNPs for 9 hrs at 100 μg/mL. Immunoblotting showed that PAH-UCNPs but not PAA-UCNPs decreased the protein level of p62, a signature of autophagosome degradation (Fig.3), which was consistent with the previous studies (Figs. 1 and 2). Notably, PAH- but not PAA-coated UCNPs suppressed mTOR phosphorylation. mTOR phosphorylation can be activated by protein kinase B (PKB/AKT) and the later also negatively modulates autophagy, we next measured AKT phosphorylation. It turned out that PAH-UCNPs inhibited AKT activation but PAA-UCNPs failed (Fig.3). These results therefore suggested PAH-UCNPs probably induced autophagy initiation by targeting the AKT/mTOR signaling pathway.
(4) PAH-UCNPs induce apoptosis in blood cancer cells
Autophagy has been showed to protect from survival threats in many cells, but more and more studies demonstrated that autophagy also leads to cell death, such as apoptosis. To find out the effects of PAH-UCNPs on final cell fate, we examined apoptotic hallmarks upon UCNP treatment. As shown in Fig.4A, both PARP and caspase-3 were cleaved by PAH-UCNPs, but not affected by PAA-UCNPs. There was more caspase-3 activated with the increase of the concentration of PAH-UCNPs (Fig.4B). These results suggested PAH-UCNPs but not PAA-UCNPs induced myeloma cell apoptosis. To find out the effects of these UCNPs on anti-cancer drugs induced apoptosis, three classic anti-myeloma drugs dexamethasone, bortezomib and doxorubicin were used to treated MM cells in the presence or absence of PAH-UCNPs. It showed that PAH-UCNPs enhanced the cleavage of PARP and caspase-3, especially in cells treated with bortezomib (Fig. 5C). However, PAA-UCNPs failed to induce apoptosis or enhance apoptosis in cells treated with bortezomib (Fig.4D). Therefore, the results above indicated that PAH-UCNPs could induce both apoptosis and autophagy, and PAA-UCNPs did not induce apoptosis or autophagy.
(5) PAH- but not the PAA-coated UCNPs induce autophagy in fresh mice granulocytes
As PAH-UCNPs could induce autophagy in human blood cancer cells, what about the effect on healthy blood cells? To answer this question, granulocytes cells were isolated from healthy mice and then exposed to PAH- and PAA-UCNPs for 9 hours at 100 μg/mL. Similar results could also be observed in mice granulocytes. As shown in Fig.5 PAH-UCNPs induced the accumulation of LC3-II and Beclin1, but PAA-UCNPs did not show any effects. These results showed that PAH-UCNPs but not PAA-UCNPs could induce autophagy in mice granulocytes, which was consistent with the effects in blood cancer cells.
Fig.1. PAH-UCNPs but Not PAA-UCNPs induce autophagy in human blood cells. (A) Multiple myeloma (OPM2, 8226) and leukemia (K562) cells were exposed to PAH- or PAA-UCNPs for 9 hours at a concentration of 100 μg/mL before analysis. The transformation of LC3-II was analyzed by western blotting using a specific antibody. (B) OPM2 cells were incubated with PAH-UCNPs at increased concentrations or extended periods as indicated. Cell lysates were then subject to immunoblotting. (C) Immunofluorescent analysis of LC3 puncta. OPM2 cells were treated with PAH- or PAA-UCNPs at a concentration of 100 μg/mL for 9 hours, then stained with specific antibody against LC3-II before being applied to confocal microscopic analysis.
Fig.2. PAH-UCNPs induce autophagy by affecting the PI3KC3/Beclin1/Atg14 complex. (A) OPM2 cells were exposed to PAH-UCNPs or PAA-UCNPs in the presence or absence of 3-MA or BafA.d. The accumulation of LC3-II was analyzed by a specific antibody. Starvation was set as a positive control. (B) OPM2 cells were treated with PAH-UCNPs or PAA-UCNPs, followed by immunoblotting against Beclin1 or Bcl-2. (C) OPM2 cells were treated with PAH-UCNPs or PAA-UCNPs at indicated concentrations for 9 hours or 100 μg/mL for indicated periods followed by immunoblotting against Beclin1. (D) OPM2 cells were treated with PAH-UCNPs or PAA-UCNPs at 100 μg/mL for indicated periods followed by immunoblotting against Atg14.
Fig.3. PAH-UCNPs suppress the AKT/mTOR signaling. OPM2 cells were exposed to PAH-UCNPs or PAA-UCNPs at a concentration of 100 μg/mL for 9 hours, followed by immunoblotting against indicated proteins.
Fig.4. PAH-UCNPs induce blood cancer cell apoptosis. (A) OPM2 cells were treated with PAH-UCNPs or PAA-UCNPs for 9 hours at a concentration of 100 μg/mL, followed by the detection of PARP and caspase-3 by immunoblotting assay. (B) OPM2 cell lines were exposed to PAH-UCNPs at an increasing concentration for 9 hours, caspase-3 activation was detected by immunoblotting. (C) OPM2 cells were exposed to anti-myeloma drugs Dex, BZ, or Dox in the presence or absence of PAH-UCNPs for 9 hours, followed by immunoblotting for the activation of PARP and caspase-3. (D) OPM2 cells were treated with PAA-UCNPs and/or BZ, PARP cleavage was detected.
Fig.5. PAH-UCNPs induce autophagy in fresh mice granulocytes. Myeloid cells were isolated from mice and then exposed to PAH or PAA-UCNPs for 9 hours at 100 μg/mL, followed by immunoblotting against Beclin1 and LC3-II.