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Antitumor effect of arsenic trioxide in human K562 and K562/ADM cells by autophagy.

Arsenic trioxide (As2O3) has been reported to have potent antitumor effects in vitro and in vivo by inducing cell death via cell cycle arrest and apoptosis in leukemia cells, but the mechanisms of As2O3-mediated cell death are not fully understood. In this study, we provided in vitro evidence that As2O3 was a potent inducer of autophagy in leukemia K562 and its drug-resistant line K562/ADM cells. As2O3 significantly activated autophagic cell death (programmed cell death type II) in leukemia cell lines. Numerous large cytoplasmic inclusions, abundant autophagic vacuoles, phagocytizing cytoplasm and organelles were observed in As2O3-treated cells using electron microscope. MDC-labeled autophagic vacuoles were observed by fluorescent inverted phase contrast microscopy and the enhanced MDC fluorescent staining was detected by flow cytometry in As2O3-treated cells. Furthermore, real-time quantitative RT-PCR revealed that the expression levels of Beclin-1 and LC3 genes, which play key roles in autophagy, increased in As2O3 treated samples than in controls, indicating that autophagy can potentially be involved in the antitumor properties of As2O3. The expression level of Bcl-2 gene, an anti-apoptotic molecule, decreased in As2O3 treated samples than in controls, suggesting that Bcl-2 may be involved in accumulating Beclin-1 and triggering autophagic cell death in As2O3-treated leukemia cells. Western blotting also showed that As2O3 up-regulated Beclin-1. Altogether, our data provide direct evidence that autophagic cell death is critical for the effects of As2O3 on acute myelogenous leukemia cells.


Introduction
Arsenic trioxide (As 2 O 3 ), exhibiting potent antitumor effects both in in vitro and in vivo, has been used successfully in the treatment of patients with newly diagnosed and relapsed or refractory acute promyelocytic leukemia (APL) Shen et al., 1997;Soignet et al., 1998). In vitro experiments have shown As 2 O 3 can exert apoptosis and induces partial differentiation effects on APL cells . In addition, it has been reported that the effects of As 2 O 3 are not confined to APL cells but can also be observed in a variety of malignant hematopoietic cells such as lymphoid (Akao et al., 1998;Hu et al., 2003), myeloid (Li and Broome et al., 1999), megakaryocytic (Lu et al., 1999), plasma cells (Rousselot et al., 1999;Gartenhaus et al., 2002) and solid tumors such as advanced hormone-refractory prostate cancer, renal cell cancer, cervical cancer and refractory transitional cell carcinoma of the bladder (Anthony, 2001). In cell based assays, As 2 O 3 can induce apoptosis effect on K562 and K562/ADM cell lines. The apoptosis mechanism may involve both the mitochondrial and endoplasmic reticulum cell death pathway. As 2 O 3 is able to reverse the apoptosis resistance in drug-resistant K562/ADM cells by modulating expression or activity of key factors associated with apoptosis induction (Jing et al., 2009;Wang et al., 2005). The mechanisms of action of arsenic trioxide in malignant cells are not well understood though extensive research has been conducted over the years.

research arTIcLe
Antitumor effect of arsenic trioxide in human K562 and K562/ADM cells by autophagy Autophagy, defined as type II programmed cell death (PCD), is independent of phagocytes and differs from apoptosis by the presence of autophagosomes, autolysosomes, and an intact nucleus in the cell (Levine and Klionsky, 2004). Studies have shown that autophagy is not only a survival response to either nutrient deprivation or growth factor but an important mechanism for tumor cell apoptosis and suppression (Gozuacik and Kimchi, 2004). More importantly, some investigations have demonstrated that the co-regulation of both apoptosis and autophagy can participate in mammalian cell death (Cheng et al., 2008). In addition, more recent work shows that As 2 O 3 can induce autophagic cell death in malignant glioma cells by up-regulation of mitochondrial cell death protein BNIP3 (Kanzawa et al., 2005); and induce autophagic cell death and apoptosis both in Molt-4 and Mutz-1 cell lines .
We report here the autophagy effect of As 2 O 3 in human leukemia K562 cells and its drug-resistant line K562/ ADM cells.

Cell line and cell culture
Adriamycin-selected and P-gp positive multidrug-resistant human leukemia cell K562/ADM and its parental K562 cells were cultured and passaged in the Central Laboratory of Medical Science, School of Medicine, Lanzhou University. The cells were maintained in RPMI-1640 medium (Gibco, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Sijiqing Biological Engineering Company Limited, Hangzhou, China), 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37°C in a 5% CO 2 incubator. The cells in mid-log phase were used for experiments. As 2 O 3 was dissolved in sterile double-distilled water at a stock concentration of 40 mg/mL, stored at 4°C. RPMI-1640 medium was used to obtain the desired concentration for experiments. 3-Methyladenine (3-MA) (Sigma, USA) was dissolved in heated sterile double-distilled water to make a 100 mmol/L stock solution and then added to the medium after heated for a final concentration of 2 mmol/L.

MDC staining of autophagic vacuoles
Monodansylcadaverine (MDC) (Invitrogen, USA) staining of autophagic vacuoles was performed for autophagy analysis as previously described (Munafó and Colombo, 2001). K562 and K562/ADM cells were divided into control (0 µmol/L As 2 O 3 treatment group), As 2 O 3 (2 µmol/L and 4 µmol/L) treatment and As 2 O 3 combined with 3-MA (Sigma, USA) treatment for experiments. The cells were incubated for 24 h and 48 h. Autophagic vacuoles were labeled with 0.05 mmol/L MDC in PBS at 37°C for 10 min. After incubation, the cells were washed three times with PBS and immediately analyzed under fluorescent inverted phase contrast microscope (Olympus, Tokyo, Japan). Fluorescence of MDC was measured at the excitation wavelength 488 nm with an emission filter at 530 nm.

Transmission electron microscope
K562 cells (2 µmol/L As 2 O 3 treated for 48 h) and K562/ ADM cells (4 µmol/L As 2 O 3 treated for 24 h) were fixed with 2.5% glutaraldehyde in 0.1 mol/L PBS (pH 7.4) for 90 min at room temperature, and post-fixed in 1% osmium tetraoxide for 30 min. After being washed with PBS, the cells were progressively dehydrated in a 10% graded series of 50-100% ethanol and propylene oxide, and embedded in Epon 812 resin. The blocks were cut into ultrathin sections with a microtome, which were then stained with saturated uranyl acetate and lead citrate. The ultrastructure of the cells was examined under a transmission electron microscope (JEM-1230, JEOL, Japan).

Flow cytometry
After being treated with As 2 O 3 , the K562 and K562/ADM cells were collected, labeled with MDC, and the fluorescence stainning of cells was analyzed by flow cytometry (EPICS XL, Beckman Coulter, USA).
β-actin was used as an internal control to evaluate the relative expressions of Beclin-1, LC3 and bcl-2. The PCR conditions were as follows: a pre-denaturing at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing/extension at 60°C for 30 s. The 2-ΔΔCT method was used to calculate the relative abundance of target gene expression generated by Rotor-Gene Real-Time Analysis Software 6.1.81. For each cDNA, the target gene mRNA level was normalized to β-actin mRNA level. Results were expressed as the ratio of normalized target gene mRNA level in cells treated with arsenic trioxide to that in cells untreated with arsenic trioxide. The experiments were performed in triplicate.

Statistical analysis
All data were expressed as mean ± SD. Statistical analysis was performed using the SPSS 16.0 for Window. One-way analysis of variance (ANOVA) was used to analyze statistical differences between groups under different conditions. p < 0.05 was considered statistically significant.

Observation of autophagic vacuolization in cytoplasm by fluorescent inverted phase contrast microscopy
MDC is a specific marker for autophagic vacuoles. When the cells were viewed under a fluorescent inverted phase contrast microscope, MDC-labeled autophagic vacuoles appeared as distinct dot like structures distributed in cytoplasm or in perinuclear space. The fluorescent density and MDC-labeled particles of K562 and K562/ADM cells were higher in As 2 O 3 treatment group than in control group (Figures 1 and 2), indicating that As 2 O 3 induces formation of MDC-labeled vacuoles. 3-MA, which is generally accepted as a specific inhibitor of autophagy, can decrease the number of autophagic vacuoles (Zhang et al., 2010). Lower fluorescent density was observed in combined 3-MA and As 2 O 3 treatment group, showing that 3-MA exerts its inhibitory effects on As 2 O 3 -treated autophagy.

Transmission electron microscopy revealed formation of autophagosomes in As 2 O 3 -treated K562 and K562/ ADM cells
Transmission electron microscopy (TEM), the standard method to detect autophagy (Mizushima, 2004), to further clarify whether the cell vacuolization induced by As 2 O 3 is involved in autophagy, we performed TEM to detect the K562 cells treated with As 2 O 3 at the concentration of 2 µmol/L for 48 h, the K562/ADM cells treated with As 2 O 3 at the concentration of 4 µmol/L for 24 h. The cells not treated with As 2 O 3 exhibited the normal ultrastructural morphology of cytoplasm, organelles and nuclei ( Figure 3). The most apparent morphological change in As 2 O 3 -treated cells was the formation of double-membranes, abundant autophagic vacuoles phagocytizing cytoplasm and organelles, such as mitochondria and endoplasmic reticulum. Giant autophagosomes filled with degraded organelles, variant mitochondria and autolysosomes were frequently observed. These results     demonstrated As 2 O 3 could induce K562 and K562/ADM cells to generate autophagy, which was consistent with the vacuolization obtained by inverted phase contrast microscopy.

The MDC fluorescent staining of As 2 O 3 -treated cells
MDC staining assay showed As 2 O 3 induced autophagy of K562 and K562/ADM cells (Figures 4-7). Flow cytometry showed the number of MDC-positive cells was 1% in K562 cells not treated with As 2 O 3 , and was 20.9% and 82.6% in K562 cells treated with As 2 O 3 at the concentration of 2 µmol/L for 24 h and 48 h; was 48.7% and 50% in K562 cells treated with As 2 O 3 at the concentration of 4 µmol/L for 24 h and 48 h (Figures 4 and 5). It showed the number of MDC-positive cells was 1.42% in K562/ADM cells not treated with As 2 O 3 , and was 10% and 33.2% in K562/ADM cells treated with As 2 O 3 at the concentration of 2 µmol/L for 24 h and 48 h; was 49.1% and 40% in K562/ADM cells treated with As 2 O 3 at the concentration of 4 µmol/L for 24 h and 48 h. The results showed the number of MDC positive K562 cells was obvious increased after 2 µmol/L As 2 O 3 treated for 48 h; and the number of MDC positive K562/ADM cells was obvious increased after 4 µmol/L As 2 O 3 treated for 24 h. As 2 O 3induced autophagy in K562/ADM cells occurred earlier than in K562 cells.

As 2 O 3 up-regulated mRNA expression of Beclin-1 and LC3 but down-regulated mRNA expression of Bcl-2 in K562 and K562/ADM cells
In order to understand the molecular mechanism underlying the autophagy induced by As 2 O 3 , real-time quantitative RT-PCR was performed to evaluate the effect of As 2 O 3 on mRNA expression of Beclin-1 and LC3, which play key roles in autophagy Kabeya et al., 2000). Real-time quantitative RT-PCR showed As 2 O 3 activated the Beclin-1 and LC3 gene expression (Figure 8). At the same time, the mRNA expression of Bcl-2 decreased (Figure 9).

Up-regulation of Beclin-1 protein contribute to autophagic cell death induced by As 2 O 3
To examine the mechanism of As 2 O 3 -induced autophagic cell death, we next performed western blot assay to evaluate the effect of As 2 O 3 on expression of Beclin-1 protein. The results showed As 2 O 3 induced increase in Beclin-1 protein expression of K562 cells, especially after 2 µmol/L As 2 O 3 treated for 48 h (Figure 10), and an obvious increase in Beclin-1 protein expression of K562/ADM cells after 4 µmol/L As 2 O 3 treated for 24 h (Figure 11), consistent with RT-PCR gene expression profile. The results showed K562 cells treated with As 2 O 3 at the concentration of 2 µmol/Lfor 48 h occurred the most apparent autophagic variability changes, and then K562/ADM cells treated with As 2 O 3 at the concentration of   4 µmol/L for 24 h resulted in the most apparent autophagic variability changes compared to control groups.

Discussion
We have demonstrated As 2 O 3 inhibited both proliferation and viability of K562 and K562/ADM cells in a dose-and time-dependent manner, in which apoptosis is identified as the main mechanism, and the drug-resistant K562/ ADM cells were more sensitive to As 2 O 3 , previously (Jing et al., 2009;Wang et al., 2005). Although much is known on the mechanism of arsenic trioxide induction of apoptosis, very little is known on the mechanism of    autophagy induction by arsenic trioxide. In this study, we directly examined the ability of As 2 O 3 to induce autophagic cell death in leukemia cell lines.
Our results demonstrated autophagy was activated with As 2 O 3 induced death of K562 and K562/ADM cells, revealing that the mechanism underlying the cytocidal action of As 2 O 3 is more complex.
While under normal conditions, autophagy contributes to the turnover of long-lived proteins and elimination of damaged or aged organelles to maintain cell homeostasis (Yang et al., 2008). Further more, autophagy is implicated in multiple pathological processes, including infection by pathogens, inflammatory bowel disease, neurodegeneration and cancer (Morselli et al., 2009). On the one hand the hypoxia-induced autophagy leads to chemoresistance of hepatocellular carcinoma cells (Song et al., 2009), whereas there is evidence that inhibition of autophagy appears to enhance the effects of BCR-ABL kinase inhibitors on Ph(+) primary chronic myelogenous leukemia stem cells (Bellodi et al., 2009). All of them indicated autophagy may act as a protective mechanism for malignant cells. On the other hand, more and more recent evidences showed autophagy plays an important role in tumor formation and suppression. Improper autophagy can result in cell death (Yang et al., 2008;Morselli et al., 2009).
Beclin-1, which maps to a tumor susceptibility locus on chromosome 17q21, is a mammalian orthologue of the yeast Apg6/Vps30 gene, is frequently monoallelically deleted in human breast, ovarian, and prostate cancers (Aita et al., 1999). Beclin-1 functions in autophagy as part of class III phosphatidylinositol 3-kinase (PI3k) complex, it binds to PI3KC3 or Bcl-X(L) to form complexes of Beclin-1-PI3KC3 or Beclin-1-Bcl-X(L) (Kihara et al., 1999). Beclin-1 gene transfer increases basal levels of autophagy and nutrient deprivation-induced autophagy in human breast carcinoma cells that lack detectable levels of endogenous Beclin-1 protein (Liang et al., 2001). Beclin-1 could be linked with HIF1a expression and poor survival of nasopharyngeal cancer patients undergoing radiotherapy and chemotherapy (Kim et al., 2008). Blocked autophagy also sensitizes cancer cells to radiation (Apel et al., 2008).Conversely, tumours with repressed Beclin-1 expression seem to bear increased inherent radio resistance because of increased antiapoptotic potential (Anai et al., 2007). In our study, the mRNA expression level of Beclin-1 and the expression of Beclin-1 proteins in K562 and K562/ADM cells was measured. As 2 O 3 treatment increased the expression of Beclin-1 gene ( Figure 8A and 8B) and Beclin-1 protein in K562 and K562/ADM cells (Figures10 and 11), especially in K562 cells treated with 2 µmol/L As 2 O 3 for 48 h, in K562/ADM cells treated with 4 µmol/L As 2 O 3 for 24 h. Indicatingthat up-regulation of Beclin-1 may contribute to As 2 O 3 -induced autophagy and play an important role in the non-apoptotic cell death induced by As 2 O 3 .
As we know, LC3, an autophagosomal ortholog of yeast Atg8, was identified as the first mammalian protein localized in the autophagosome membrane. It is considered as an autophagosomal marker. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes (Tanida et al., 2004). Thus detecting LC3 gene by RT-PCR and LC3 protein by immunoblotting or immunofluorescence has become reliable methods for monitoring autophagy and autophagy-related processes, including autophagic cell death.
We detected the mRNA expression of LC3 gene ( Figure  8C and 8D), including LC3I and LC3II, suggesting that arsenic trioxide activates expression of the LC3 gene, which acts later to complete the autophagosome formation. Prompting that not only LC3 protein involves in the process of autophagy, but also the LC3 gene.
The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin-1, which not only regulates apoptosis, but also controls nonapoptotic programmed cell death that depends on the autophagy genes (Shimizu et al., 2004). Down-regulation of Bcl-2 in Molt-4 cells exposed to As 2 O 3 , suggesting Bcl-2 may contribute to autophagy induced by it . The interaction between Bcl-2 and Beclin-1 in the endoplasmic reticulum blocks a signal that is essential for the formation of the Beclin 1-Vps34 autophagy-promoting complex (Pattingre and Levine, 2006). In this study, we found when autophagyis induced, the mRNA expression of Bcl-2 decreased both in K562 and K562/ADM cells (Figure 9).
We found that the obvious autophagic changes induced by As 2 O 3 in K562 and K562/ADM cells occurred at different dose and time. and the autophagy of K562/ ADM cells displayed earlier than that of K562 cells. These may partly explain why K562/ADM cells are more sensitive to arsenic trioxide-induced cell death (Jing et al., 2009;Wang et al., 2005).
In conclusion, As 2 O 3 is a potent antitumor agent by inducing cell autophagy besides apoptosis. The relationship between apoptosis and autophagy induced by arsenic trioxide remains unclear, and further study is required to understand the difference of molecular interactions in arsenic trioxide-induced autophagy between drug-sensitive and -resistant leukemia cells, and the relevance of arsenic trioxide-induced autophagy and apoptosis in leukemia.

Declaration of interest
This work is supported by Technology research and developmental projects of Gansu Province, China, No. 1105TCYA003. This study was funded by the 'Stichting Instituut Gak' . The authors report no conflicts of interest that are directly relevant to the content of this article.