Biol Trace Elem Res (2014) 161:3–12 DOI 10.1007/s12011-014-0068-7

Research Advances on Apoptosis Caused by Quantum Dots Qingling Zhan & Meng Tang

Received: 10 May 2014 / Accepted: 8 July 2014 / Published online: 27 July 2014 # Springer Science+Business Media New York 2014

Abstract Recently, quantum dots (QDs) have been widely applied in biological and biomedical fields such as cell labeling, living tissue imaging, and photodynamic therapy because of their superior optical properties. Meanwhile, the potential biological negative effects and/or toxic effects of QDs have become increasingly important, especially the cytotoxicity caused by QDs. One of the common cytotoxicity when living organisms are treated with QD is apoptosis, where many attempts have been made to explain the mechanisms of apoptosis caused by QDs’ use. One of the mechanisms is the production of cadmium ion (Cd2+) and reactive oxygen species (ROS). Excess generation of ROS will result in oxidative stress that would mediate apoptosis. Furthermore, the activation of cell death receptors and mitochondria-dependent such as B cell lymphoma 2 (Bcl-2) family and the caspase family could onset apoptosis. Signal transduction such as some classical signal pathways of PI3K-AKT, NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE), mitogenactivated protein kinases (MAPKs), and nuclear factor kappa B (NF-κB) also plays an important role in the regulation of apoptosis. Several ways to reduce the apoptotic rate have been introduced, such as surface modification, controlling, the dose, size, and exposure time of QDs as well as using antioxidants or inhibitors. In this review, we attempted to review the most recent findings associated with apoptosis caused by QDs Q. Zhan : M. Tang Key Laboratory of Environmental Medicine and Engineering, Ministry of Education; School of Public Health & Collaborative Innovation Center of Suzhou Nano Science and Technology, Southeast University, Nanjing 210009, Jiangsu Province, People’s Republic of China Q. Zhan : M. Tang (*) Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing 210009, Jiangsu Province, People’s Republic of China e-mail: [email protected]

so as to provide some guidelines for a safer QD application in the future. Keywords Quantum dots . Apoptosis . Mechanism . Biological negative effects . Toxicity living organisms . Nanotoxicology

The Chemical and Physical Properties of QDs Quantum dots (QDs), also known as semiconductor nanocrystals, is a type of nanomaterial that has been extensively applied in biology and medicine. Just like many natural or synthesized nanoparticles, synthesized QDs have their unique physical and chemical properties that are determined by mainly two factors: the crystal structure of the metal core and the quantum size confinement. A typical QD is made up of a crystal metal core and a “cap” or “shell” that can blunt the nuclear material properties and improve the QDs’ biocompatibility. The core is composed of semiconductor QDs, heavy metals, magnetic transition metals, and other kinds of metallic complexes. In addition, group III–V series QDs are made up of nonmetal cores, such as indium phosphide (InP), indium arsenide (InAs), gallium arsenide (GaAs), and gallium nitride (GaN), and group II–IV series QD cores are made of zinc sulfide (ZnS), zinc selenide (ZnSe), cadmium selenide (CdSe), and cadmium telluride (CdTe) [1]. Furthermore, synthetic routes to new, heavier structures (e.g., CdTe/CdSe and CdSe/ZnTe) and hybrids composed of lead selenium (PbSe) have also been established [2]. The surface of QDs is either functionalized with organic molecules such as –COOH, –NH2, and –OH that can improve QDs’ biocompatibility [3] or specific functional groups such as captopril (a kind of antihypertensive drug) that can reduce the toxicity of QDs and the effects of hypertension [4].

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In dimension, the diameter of a QD is less than or close to the exciton Bohr radius (diameter less than 10 nm); therefore, it is a tiny dot object and has a quasi zero-dimensional nanostructure. As internal electrons’ movement in QDs is limited in all directions, the quantum confinement effect is prominent [5]. At nanoscale level, the small particle size of QDs not only allows the quantum confinement effect, but also the unique surface effect [6]. On the other hand, the number of atoms on QDs’ surface increases rapidly with the decrease of QDs’ particle size. These surface atoms, due to lacking adjacent atoms, will generate many dangling bonds that are prone to combine with other atoms to become saturated and stable. Hence, the surface of the QDs has a high chemical reactivity.

Applications of QDs and Their Potential Toxicity QDs have unique optical and electronic properties with broad absorption and narrow emission ranges [7], which can be applied in a variety of industrial and biomedical areas, such as tracking of macromolecules inside cells and tissues and labeling of organelles and cells [8–11]. The biosafety of QDs have attracted attentions of researchers worldwide because QDs can be unintentionally exposed to human at workplace or during the end product use via inhalation, dermal absorption, or gastrointestinal tract absorption [12], which may cause diverse toxic effects. At cellular levels, QDs promote the formation of ROS by depleting cellular antioxidants, which may lead to mitochondrial dysfunction, cell apoptosis induction, intracellular calcium signaling channel disruption, DNA damage, DNA repair inhibition, and cell death induction [13]. At tissue and organ levels, the chronic intake of QDs could release Cd2+, which may result in inflammation and fibrosis and eventually organ dysfunction [13–15]. In this review, we focused on apoptosis, one of the cytotoxicity caused by QDs and aim to summarize recent findings on the precise regulatory mechanisms underlying the above mentioned effects (Fig. 1). We also attempted to gather various ways to hinder cell apoptosis or reduce the apoptotic rate.

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fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope.

Apoptosis Induced by Cadmium Ion (Cd2+) Most cores of QDs applied in biological and biomedical lines of research are composed of Cd2+ that was known to be a toxicant that could induce apoptosis [16, 17]. Yu et al. [18] investigated the effects of cadmium chloride (CdCl2) on hepatocellular apoptosis in rats at the doses of 5, 10, and 20 mμmol/kg by using TdT-mediated dUTP nick end labeling (TUNEL). The results suggested that CdCl2 could induce hepatocellular apoptosis to in vivo rats at certain levels. Similar findings were reported by Tzirogiannis et al. [19]. However, comparing with necrosis, the apoptosis is not a completely bad thing sometimes. In the research of Habeebu et al. [20], mice were injected with 5–60 mμmol/kg i.p. of Cd. After 1.5– 48 h, their livers were removed and examined by light microscopy. Cd induced both a time- and dose-dependent increase in apoptotic index, mitotic index, and severe necrosis. However, it was very interesting to observe that cadmiuminduced liver injury did not involve inflammation at any time point. Apoptosis seemed to be a major mechanism for the removal of damaged cells, and it preceded so to necrosis constituting the major type of cell death in nonparenchymal liver cells. Apart from in vivo studies that measured Cd or Cd2+-induced apoptosis, several in vitro studies also demonstrated the cytotoxic effects of Cd or Cd2+ that cause apoptotic cell death in several cell lines (e.g., hepatic ones). Hart et al. [21] investigated the mode of cadmium (Cd2+)-induced cell death in a rat lung’s epithelial cell lines. Cells that have grown to near confluence were exposed to 0–30 μM CdCl2 for 0– 72 h. Subsequent phase contrast microscopy and fluorescent nuclear staining showed that Cd has induced morphological alterations in lung epithelial cells which were characteristics of apoptosis. Li et al. [22] and Oh et al. [23] also reported similar results. Although the above studies indicated that Cd or Cd2+ might play an important role in apoptosis, the particular molecular events which lead to cell apoptosis still remain fairly unclear.

Biological Characteristics of Cell Apoptosis Apoptosis or programmed cell death is a particular form of cell death where the cell undergoes intentional suicide. It is mediated by the production of ROS, specific receptors, and signal transduction. Characteristics of apoptosis and the morphological alterations caused by QDs in cells can be shown by phase contrast microscopy and fluorescent nuclear staining. These characteristic changes are often cited as the apoptotic detection index, which include cell shrinkage, detachment of the cell from its neighbors, cytoplasmic and chromatin condensation, and

ROS-Mediated Apoptosis QDs could induce the production of ROS mainly through interactions with cellular molecules, where QDs act as photosensitizers and transfer energy to these molecules [24]; for cadmium-based QDs releasing cadmium ion (Cd2+) can led to the production of ROS, too. ROS generated in cells that include singlet oxygen (1O2), superoxide anion (O2−·), hydroxyl radicals (OH), and hydrogen peroxide (H2O2) could result in oxidative stress and affect cellular signaling cascades

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Fig. 1 Molecular events

that control different cellular processes leading to cell damage and triggering apoptosis [25]. Similarly, in the experiment of Oh et al. [26] where HepG2 cells were treated with different concentrations of Cd, a rapid and transient ROS generation has triggered Cd-induced apoptosis. ROS performs an important factor in early stages of apoptosis, where it induces the depolarization of the mitochondrial membrane which eventually results in an increase in ROS as well as other pro-apoptotic molecules levels in the cytosol [27, 28]. Luo et al. [29] suggested that cadmiumcontaining QDs caused an increase of intracellular ROS levels in mouse renal adenocarcinoma (RAG) cells and induced autophagy and subsequent apoptosis at 6 and 24 h after treatment, respectively. Lovric et al. [30] showed that “naked” QDs induced damage to the plasma membrane, mitochondrion, and nucleus, leading to cell apoptosis or cell death. CdTe QDs would induce cell death by nonclassical apoptosis initiated by ROS detectable in live human breast cancer (MCF-7) cells. Hsu et al. [31] demonstrated that dissolved zinc ions (Zn2+) in the medium raised the cellular uptake of ZnO QDs

and ROS, inducing cell apoptosis. Therefore, ROS are important players in mediating QD-induced cell apoptosis. Excess generation of ROS in cells leads to oxidative stress, which in turn induces a cascade of reactive oxygen detoxification systems. If the balance tips are in favor of pro-oxidant stress, antioxidant defenses would collapse and result in cell apoptosis. Buttke et al. [32] demonstrated that oxidative stress played an important role in triggering apoptosis. Furthermore, Li et al. [33] elucidated the relationship between Cd2+ and oxidative stress with experiments of biosurfactant stabilized CdS QDs (bsCdS QDs). They observed that Cd2+ ions could replace iron and copper in various cytoplasmic and membrane proteins (e.g., ferritin and apoferritin) in the cellular environment, which consequently increased the amount of free or chelated copper and iron ions that participated in oxidative stress via Fenton reactions. Their results suggested that bsCdS QDs have the capacity to generate free radicals indirectly and induce oxidative stress and apoptosis by releasing Cd2+ in cells. Similarly, Nguyen et al. [34] found that CdTe QDs elicited oxidative stress by generating ROS, resulting in

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apoptosis in hepatocellular carcinoma HepG2 cells in a timeand dose-dependent manner. While QDs could elicit a series of oxidative damage, they inevitably encounter a number of defense mechanisms that deal with ROS generation, which may eliminate, sequester, or dissolve them in cells [35]. As shown in Fig. 2 [36], phase II antioxidant enzymes are induced via transcriptional activation of the antioxidant response element by Nrf-2 to restore cellular redox homeostasis at a lower level of oxidative stress (tier 1). At an intermediate level of oxidative stress (tier 2), activations of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) cascades induce pro-inflammatory responses. At a high level of oxidative stress (tier 3), perturbations of the mitochondrial PT pore and disruptions of electron transfer result in cellular apoptosis or necrosis [36]. Apoptosis does not seem to be beneficial to normal cells, but it may be a different story for cancer cells. If ROS can be selectively over-produced in tumor cells that induce apoptosis, they may exert remarkable antitumor potentials through their reactions with vital cellular targets [37]. This strategy of cancer treatment by selectively generating excess ROS in tumor cells to achieve maximum tumor killing without affecting normal tissues [38] has been termed as “oxidation therapy.” Singh et al. [39] synthesized highly stable and surface protected CdS QDs (denoted as “bsCdS QDs”) using biosurfactant and demonstrated that bsCdS QDs induced apoptotic cell death in human prostate cancer lymph node carcinoma of the prostate (LNCaP) cells. This suggests that the oxidation therapy may kill cancer LNCaP cells via inducing apoptotic cell death. Similarly, Zhao et al. [40] combined quantum dots (QDs) with glycyrrhizic acid (GA) in the presence of β-cyclodextrin (β-CD) to prepare β-CD/GA-functionalized QDs, which led to improved antitumor activity and apoptosis in hepatocarcinoma cells. Apoptotic response Fig. 2 The hierarchical oxidative stress model (glutathione/ oxidized glutathione (GSH/ GSSG) can respond to oxidative stress conditions)

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would be induced by β-CD/GA-functionalized QDs in a timeand dose-dependent way, but not by L-Cys-β-CD-functionalized QDs or GA alone. These studies suggested that β-CD/ GA-functionalized QDs have therapeutic potential against cancers when they were modified with proper functional groups. Further lines of research of this kind will be valuable to antitumor therapies for patients.

The Extrinsic Apoptotic Pathway—Onset of Apoptosis Apoptosis occurs in cells via two main routes, the mitochondria-dependent way (the intrinsic pathway) or the activation of death receptors (the extrinsic pathway) [41]. Fas-mediated cell death and the activation of caspase-8 have been suggested to be related to extrinsic apoptosis. Nguyen et al. examined the Fas level after treating HepG2 cells with CdTe QDs and CdCl2 and confirmed the expressions of Fas and caspase-8 to be markers of early apoptosis [34]. Compared to the control, treatment of CdTe QDs and CdCl2 induced a marginal increase in Fas level as well as increased caspase-8 activity in HepG2 cells, indicating that CdTe QDs induced apoptosis at early phase in HepG2 cells via the extrinsic pathway. The results also demonstrated that there is a strong relationship among Cd2+, Fas level, caspase-8, and apoptosis. Choi et al. [42] also found that CdTe QD-induced toxicity involved upregulations of the Fas receptor, leading to impaired neuroblastoma cell functions and causing cell apoptosis via the extrinsic pathway. Both Fas or tumor necrosis factor (TNF) could induce cellular apoptosis. Luo et al. [43] reported that the apical caspase was activated by cell surface death receptors such as Fas and TNF. Tumor necrosis factoralpha (TNF-α) or Fas ligand bound to their receptors lead to the activation of the protease caspase-8 which either directly

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cleaves and activates the effector caspases or indirectly activates the downstream caspases through the cleavage of BH3only protein Bid. Therefore, TNF-α or Fas relays an apoptotic signal from the cell surface to mitochondria. Cadmium, as a kind of source of core for QDs, is correlated with Fas, and the production of ROS might be attributed to Fas. Preliminary validation of this hypothesis comes from findings reported by Devadas et al. [44] that Fas crosslinking induced rapid generation of ROS (within 15 min) well before the appearance of characteristic apoptotic changes. Jayanthi et al. [45] demonstrated that ROS can activate death receptor Fas derived from the mitochondria that could induce apoptosis in the cell model. Denning et al. [46] suggested that oxidative stress caused by H2O2 induced an increase in messenger RNA (mRNA) levels and proteins for both Fas and FasL in the Mode-K intestinal epithelial cell line. Oh et al. [26] reported that pretreatment of an antioxidant N-acetyl cysteine (NAC) could affect Fas protein levels, confirming that Cd-induced ROS generation is able to modulate Fas expression as the initial signal in Cd-induced apoptosis. Furthermore, NAC was shown to completely inhibit the Cd-induced apoptosis pathway in its early stages via upregulations of the catalase.

The Intrinsic Apoptotic Pathway—Onset of Apoptosis Bcl-2 Family Inductions of the intrinsic pathway involve decreased antiapoptotic signals such as B cell lymphoma 2 (Bcl-2) and translocations of pro-apoptotic signals to mitochondria such as BCL2-associated X protein (Bax) and Bak. Bcl-2 is an important inhibitor of apoptosis. Its overexpression blocks translocation of cytochrome c from mitochondria into cytosol, thus preventing cells from apoptosis [47]. Conversely, the overexpression of Bax and its translocation to mitochondria has been shown to promote the release of cytochrome c and other apoptosis-inducing factors from the mitochondria to the cytosol, triggering subsequent activations of procaspase-9 and downstream apoptotic effectors [48]. Nguyen et al. [34] showed that hepatocellular carcinoma HepG2 cells exposed to CdTe QDs led to a decrease in Bcl-2 levels and an increase in mitochondrial Bax levels, suggesting the induction of the intrinsic apoptotic pathway. The release of cytochrome c from mitochondria to cytosol confirmed the effects of QDs on cellular Bcl-2 protein members. Kong et al. [49] found that CdSe-core QDs exhibited high cytotoxicity and caused apoptosis and necrosis of JB6 (skin cells of mice) cells. After JB6 cells were treated with different concentrations of CdSe coreshell QDs for 24 h, Western blot analysis showed activations of pro-apoptotic factors including Fas, Bax, and Bid. The Caspase Family The caspase family is associated with the above-mentioned intrinsic apoptotic pathway. Lamkanfi et al. [50] demonstrated that the mitochondrial (intrinsic)

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pathway of apoptosis began with the permeabilization of the mitochondrial outer membrane. They suggested that caspase7 was redundant with caspase-3 because these related cysteine proteases shared an optimal peptide recognition sequence and had several endogenous protein substrates in common. Moreover, both caspases were proteolytically activated by the initiator caspase-8 and caspase-9 during cell death receptor and DNA-damage-induced apoptosis. Lakhani et al. [51] reported that both caspase-3 and caspase-7 were important mediators in the mitochondrial events of apoptosis. Based on these observations, Touseef et al. [52] explored the mechanisms of apoptosis induced by CuO QDs. They cultured the C2C12 cell line, evaluated the cell viability, and checked the expression of candidate genes (caspase-3 and caspase-7). The results showed that CuO QDs induced toxicity in mouse C2C12 cells in a dose-related pattern and caspase-3 and caspase-7 increased significantly in the exposed cells. The upregulated expression patterns of caspase-3 and caspase-7 in exposed mouse myoblast cells also suggested that these proteins could be excellent molecular biomarkers to assess the genotoxicity of QDs. On the other hand, the mitochondrion-mediated intrinsic apoptotic pathway also includes the production of cytochrome c and the activation of caspase-9. Gen et al. [53] used Western blotting to explore the mechanism of anticancer activity after cell treatment by CdTe QDs with anticancer drug daunorubicin (DNR). They found multidrug-resistant human hepatoma HepG2/ADM cells apoptosis with a rapid induction of cytochrome c, cleaved caspase-9 and caspase-3 activities, as well as the activation of stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Similarly, Chan et al. [54] showed that CdSe-core QDs could induce apoptosis through caspase-9- and caspase-3-mediated apoptotic pathways in IMR-32 cells. There are two types of apoptotic caspases: initiator caspases and effector caspases. Initiator caspases (e.g., caspase-8) cleave inactive pro-forms of effector caspases and activate them. Effector caspases (e.g., caspase6), in turn, cleave other protein substrates and induce the apoptotic process. Kong et al. [49] observed the activation of full length and cleaved caspase-8 and examined possible involvement of caspase-3, caspase-6, and caspase-9 in the process of apoptosis induced by CdSe QDs. Their results indicated that CdSe QDs induced only a slight activation of full length and cleaved caspase-6. In other words, CdSe QDs could induce cell apoptosis through enhancing the expression of these pro-apoptotic regulatory factors.

Signal Transduction and Apoptosis The Nrf2-ARE Signal Pathway Motohashi et al. [55] once reported that NF-E2-related factor 2 (Nrf2) controlled the transcription of target genes by binding to the antioxidant

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response element (ARE) located at the enhancer regions of the genes, giving rise to regulations against xenobiotic and oxidative stresses that could induce cell apoptosis. Circu et al. [56] also suggested that the activation of p38 MAPK affected the regulation of downstream transcription factors, such as p53 and Nrf2, and subsequently controlled the downstream pro-apoptotic and antiapoptotic gene expressions in response to many extracellular stimuli. In the experiment of Zhao et al. [57], 2 μg/ml QDs and/or 2.5 or 10 μg/ml Cu2+-activated Nrf2, with an increase of protein expression in L02 cells (normal human liver cells) cytoplasmic protein lysate and a translocation to the nucleus. The expression of Nrf2 levels in whole nucleus lysates of L02 cells treated with 2.5 μg/ml Cu2+ and 10 μg/ml Cu2+ was significantly enhanced by 60 and 70 % compared to control via the Western blot assay. Cu2+ plus QDs increased the protein level of Nrf2 by 30 and 86 % compared to Cu2+ alone at concentrations 2.5 and 10 μg/ml, respectively. The findings indicated that QDs and/or Cu2+induced toxicity could enhance glutathione S transferase (GST) that take part in the protection of cellular damage induced by oxidative stress via the Nrf2/ARE pathway. This suggests that cell apoptotic and antioxidant responses that are elicited by oxidant stress in hepatic cells are possibly an adaptive mechanism. He et al. [58] provided a mechanistic transcriptional model in which Cd-activated Nrf2 through a metal-activated signaling pathway involving a dynamic interplay between ubiquitination/deubiquitination and a complex formation/dissociation of Nrf2. Their results suggested that the protective effect of Nrf2 against metal toxicity helps us to determine the involvement of QD- and Cu2+-induced activation of Nrf2. The MAPK Signal Pathway During apoptosis, a series of cell signal pathways are activated, where lots of genes and proteins are involved. ROS are closely correlated with apoptosis and the proliferation of cells through the activation of mitogenactivated protein kinase (MAPK) cascades. MAPKs are stress-sensitive kinases and are involved in death by apoptosis of cells with oxidative stress [59]. The MAPK signal pathway consists of the extracellular signal-regulated kinase (ERK), the p38 MAPKs (p38), and the c-Jun N-terminal kinase (JNK) signal pathways that are important in regulating essential cellular events such as proliferation and apoptosis. The Ras→ERK-mediated survival signaling pathway is known to protect cells from apoptotic triggers [60]. Chan et al. [54] examined the effects of CdSe-core QDs on components in this pathway. Immunoblotting revealed a dose-dependent decrease in Ras and Raf-1 protein’s expressions in human neuroblastoma cells (IMR-32 cells) treated with CdSe-core QDs but not in cells treated with ZnS-coated QDs. The phosphorylated forms of ERK-1 were also detected using immunoblotting. On the other hand, the ERK-2 proteins had an approximately fourfold decrease in activations of these kinases in CdSe-core QD-

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treated IMR-32 cells. These results implicated that the decreased ERK-1/2 activity induced by CdSe-core QDs was mediated by reduced expression of the upstream enzymes, Raf-1and Ras. Previous reports have indicated that heat shock protein 90 (HSP90) prevents proteasome-mediated degradation of several signaling proteins, including Raf-1 [61]. Further analysis of CdSe QDs treatment on HSP90 protein expression in IMR-32 cells revealed that HSP90 was downregulated in cells treated with CdSe QDs but not ZnS-coated QDs. These data suggest that CdSe QD-induced apoptosis could be mediated by suppressing the HSP90 expression, which triggers the downregulation of Ras and Raf-1 and subsequent decrease in ERK 1 and ERK 2 activities. In the same experiment, pretreatment with SP600125 (a specific JNK inhibitor) on CdSe QD-treated IMR-32 cells reduced CdSe QD-stimulated JNK activity in a dose-dependent manner but had no effect on the expression levels of JNK proteins. The researchers also found that the inhibition of JNK activity due to SP600125 has significantly prevented mitochondrial membrane potential (MMP) losses and apoptosis in treated cells. These findings suggested that JNK activity is required for loss of MMP and subsequent apoptotic biochemical changes during apoptosis induced by CdSe QDs. Furthermore, pretreatment of cells with U0126 (a specific inhibitor of MEK1) enhanced CdSe QDs and induced the inactivation of ERK-1/2 which accelerated apoptosis in treated cells. In conclusion, CdSe QDs could induce apoptosis through JNK activation of some mitochondrial-dependent apoptotic processes and inhibition of these survival signaling components such as ERK in IMR-32 cells. Lu et al. [62] also showed that JNK activation was required for CdSe QDs and it could induce apoptosis in human osteoblast cells. To the contrary, Nguyen et al. [30] reported that CdTe QDs would activate MAPK signaling pathways in hepatocellular carcinoma HepG2 cells by increasing in phosphorylation levels of JNK, p38, as well as an increase in Erk1/2 which was contrary to the report from Chan and colleagues who showed that a decrease in Erk level caused by their test QDs resulted in inhibition of Ras to Erk survival signaling. The different findings might be due to differences in the test cell lines, as suggested in previous literature (e.g., Cd-induced Erk activation cell typedependent) [63]. The NF-κB Signal Pathway Nuclear factor kappa B (NF-κB) is necessary for the transcription of many key mediators in apoptotic pathways [64]. Amelia at al. [65] investigated the influence of 15-nm CdSe/ZnS–COOH QD nanocrystals (QDs) on the cell density, viability, and morphology QU exposure in human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) at two time points (8 and 48 h). They detected the induced modulation of genes (CASP1, ADORA2A ) as well as the inflammatory, proliferation, and apoptopic responses, for example, perturbed cytosolic levels

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of NFκB1 mRNA and message levels of NFκB2 that went down at both 8 and 48 h and the inhibitory NFκBIA that went up at both 8 and 48 h. (what happens with IL-1B). Stronger upregulation of IL-1β was observed at 48 h, especially at the 30-nM concentration. The TNF-α expression was slightly induced at a later time point when compared to unexposed cells. NF-κB responds to and induces IL-1β as well as death receptors TNF-α. While the ultimate gene targets of NF-κB are diverse, its activation has been shown to block apoptosis [66, 67]. Conversely, a suppression of nuclear NF-κB can result in TNF-α-induced apoptosis. Western blot results indicated that while cytosolic NF-κB remained unchanged with QD exposure, nuclear levels decreased over time. Secondary blotting for lamin, a constitutive nuclear protein, revealed that the 48-h samples exhibited a more concentrated nuclear constitution, suggesting an even stronger suppression than what was shown in the nuclear fraction blot. Probing for NF-κB (p65) in total cell lysates resulted in a fairly consistent degradation of NF-κB levels with QD treatments. These findings help to explain why apoptotic TNF-α was having increased expression over time. Activated NF-κB could restrict the actions of TNF-α in the cell and demonstrated that NF-κB signal pathway took part in the process of apoptosis and was closely related with apoptosis (to review, the article entitled “The nuclear factor NF-κB pathway in inflammation (Laurence, T.)” published in the journal CSH Perspectives in Biology, vol. 1 (6) (2009)).

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taking part in mitochondrial functions were upregulated in cells exposed to QDs. Ca2+ Signaling The involvement of Ca2+ signaling in apoptosis has been suggested in a number of recent studies. Pu et al. [70] reported that a cytosolic Ca2+ signal was involved in regulating UV-induced apoptosis in HeLa cells. They also found that the Ca2+ signal occurred at the upstream of the cytochrome c release from mitochondria. Liu et al. [71] treated L929 cells with CdTe QDs and observed cell apoptosis. The intracellular calcium levels were determined using the fluorescence Ca2+ indicator, fluo-3/AM, and analyzed by confocal microscopy. The results suggested that the fluorescence intensity was dose-dependent. The presence of QDs elevated intracellular calcium levels. As shown in the 20 and 40 μg/ml 3.5nm CdTe treatments, the fluorescence intensity increased and was significantly different to the control group, indicating that the acute application of CdTe QDs could increase intracellular calcium levels when apoptosis occurs. Overall, Ca2+ signaling is closely related to cell apoptosis.

The Way to Reduce the Rate of Apoptosis

Other Factors Related to Apoptosis

Taking into account of the wide-range use of QDs and their potential toxicity, many attempts have been made to reduce QD toxicity, particularly apoptosis. Factors that could influence nanoparticle internalization and the consequent cytotoxicity include QD core and surface compositions, the treatment time and dose of QDs, as well as the external environment of QDs [72, 73].

Genetic Expressions of Apoptosis Regulations of genes are known to play crucial roles in cellular apoptosis processes except for the death receptor and the caspase family. Amelia et al. [65] reported that the gene for the adenosine receptor (ADORA2A) had implications in programmed cell death [68] and could be upregulated by CdSe/ZnS–COOH QDs. As mentioned earlier, TNF binds its receptor ligand to elicit the extrinsic apoptotic pathway. The upregulation of TNFRSF1A suggests this apoptotic pathway as a common response to the CdSe/ZnS–COOH QD exposure. The TNF receptor superfamily member 1A (CD120a) is a receptor for TNF-α. The modulation of NLRC4, CASP1, and CASP4 implicated that apoptosis was induced. Genes CASP1 and ADORA2A were connected with an apoptotic response, as well as gene CASP9. Nagy et al. [69] investigated the upregulation and downregulation of genes by real-time PCR analysis and explored the mechanisms of CdSe QDs driving normal human bronchial epithelial (NHBE) cell death, mitochondrial function, and inflammation. The study discovered that gene CASP9 was involved in apoptosis and that genes CYP1A2 and UCP1

Surface Modification Surface modification is a frequently used approach to reduce the toxicity of QDs. As we know, unmodified cadmium telluride quantum dots (CdTe QDs) can induce cell apoptosis, but Qu et al. [74] showed that wellmodified CdTe QDs with polyethylene glycol-conjugated amine particles could exert robust inhibition on cell proliferation of J744A.1 macrophages, irrespective of apoptosis. Not only polymer-modified QDs but also the gels could avoid apoptosis. In the study of Prasad et al. [75], the viability, cytotoxicity, and apoptosis caused by red and orange QDs of gel and nongel types at different time periods were analyzed, and the effects of QD exposure on PC12 cells before and after the neurite was grown were discussed. The ApoTox Triplex assay showed the apoptosis of PC12 cells treated with NGF and exposed to red and orange QDs of gel and nongel types along with controls. Researchers also found the apoptosis of cells that were treated with red and orange gel and nongel QD types after neurite had been grown for 10 days. Overall, nongel QDs induced more apoptosis than gel QDs, which suggested that the gelatine capping present on the surface of

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QDs acts as a barrier toward the leaking of toxic ions from the core QDs. The apoptosis rate of cells treated with QDs modified with hydrophilic groups could also be reduced. Yuan et al. [76] explored the rate of cellular apoptosis, where human A549 lung carcinoma cells and human neural glioma C6 cells were treated with three kinds of graphene quantum dots (GQDs) with different modified groups (NH2, COOH, and CO–N (CH3)2, respectively) and were then evaluated by flow cytometry analysis. The results suggested that the three modified GQDs showed low cytotoxicity and excellent biocompatibility even when the concentration of decorated GQDs reached 200 μg/ml, which was a seriously damage dose for undecorated GQDs. Dose, Size, and Exposure Time Dose, size, and exposure time are considered to be the key parameters determining the toxic level of an individual QD. Liu et al. [71] have tried to determine the threshold dose for reduced or eliminated CdTeinduced toxicity in L929 cells by controlling the particle size and exposure dose of QDs. They established a cellular model of acute exposures to CdTe QDs, where cells were exposed to different concentrations of CdTe QDs (2.2 and 3.5 nm) followed by illustrative cytotoxicity analysis. They found that low concentrations of CdTe QDs (under 10 μg/ml) promoted cell viability and had no obvious effect on the rate of cell apoptosis, intracellular calcium levels, and changes in mitochondrial membrane potential, while high concentrations significantly inhibited cell viability. It was also demonstrated in this study that the rate of apoptosis induced by 2.2-nm QDs was higher than that by 3.5-nm QDs, suggesting that particle size of QDs could influence the rate of cell apoptosis. The findings agree with previous lines of research, where the observed low concentration exposure effects were dependent on QDs’ dose and exposure time [17, 77–79]. Other Influence Factors 3-Methyladenine (3-MA) is a kind of cell autophagy inhibitor. Jing et al. [80] reported that cell autophagy inhibitor 3-MA could reduce cell apoptosis at low concentrations of QDs and plays a key role in QD-mediated cytotoxicity. The research also found that the percentage of cell apoptosis decreased by15 % from 0 to 12 mM 3-MA at QD concentration of 40 μg/ml. Prasad et al. [75] proved that bsCdS QDs could elicit cell apoptosis. They also examined the apoptosis in the presence of N-acetyl cysteine (NAC), which is known as a kind of antioxidant. Results showed that upon removal of ROS by NAC, the proportion of bsCdS QD-treated cells staining positive for PI/or sub-G0/G1 content was significantly reduced from 32 to 5 %. Therefore, NAC can protect prostate cancer LNCaP cells against ROS through reducing the rate of apoptosis.

Zhan and Tang

Outlook A number of important factors that can induce cell apoptosis have been summarized in this review including the production of Cd2+ and ROS, oxidative stress, cell death receptor, the mitochondria-dependent way, and signal transduction. Strategies to reduce apoptosis rate have also been introduced, e.g., surface modification; controlling the dose, size, and exposure time of QDs; and the use of antioxidant or inhibitor. However, the specific mechanisms involved in these influential elements are still unclear. For future evaluations of QD-induced toxicity, it is essential to develop evaluation methods capable of accommodating QDs’ inherent characteristics and considering the following aspects. Firstly, QDs may cause apoptosis via special pathways because of their smaller particle size and singular physicochemical and optical characteristics. Secondly, the elucidation of QD-specific mechanisms of cellular oxidative stress and apoptosis is hampered by QD-specific physicochemical characteristics such as Cd2+ ion release and agglomeration or aggregation, all of which could influence the production of ROS, oxidative stress, the activation of cell death receptor, and kinds of signal pathways sequentially. Thirdly, numerous studies have been published with regard to QD-induced apoptosis, but the data among studies especially quantitative data such as half-maximal inhibitory concentration (IC50) values, the cell viability experiment are not truly comparable. That is because each research group has employed independent experimental designs including the preparation of QDs and the procedures related to the surface modification and dispersion of QDs, and each QD type is a unique nanoparticle, which needs to be assessed individually [81]. To sum up, it is challenging but very important to standardize and harmonize experimental procedures of QDs. We hope that more suitable, practical, and better quality QDs could be prepared and, in the future, applied in the diagnostic and therapeutic applications of various human diseases.

Acknowledgment The project was supported by the National Natural Science Foundation of China (30972504, 81172697, and 81302461). This work was supported by the National Important Project on Scientific Research of China (No. 2011CB933404). Conflict of Interest There is no conflict of interest for all authors.

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