Abstract

Introduction: The therapeutic effect of arsenic trioxide (ATO, As2O3) has been investigated for many years. However, the precise molecular mechanisms underlying the antitumor activity of ATO are still not fully understood, but seem to depend on cell types, dosage, and duration of exposure. The purpose of this study was to assess the actin cytoskeleton rearrangement during the cell death process induced by arsenic trioxide in the CHO AA8 cells. A better understanding the mechanisms of ATO-action is likely to lead to more rational use of this drug either as monotherapies or in combination with other anticancer agents.Material and methods: The effect of ATO on actin cytoskeleton was studied in Chinese Hamster Ovary AA8 cell line. Actin was visualized by fluorescence microscopy and phalloidin conjugated to Alexa Fluor® 488. Morphological and ultrastructural alterations in the CHO AA8 cells were evaluated by using light and electron microscope, respectively. For quantitative measurement of cell death, Annexin V-Alexa Fluor® 488 and Propidium Iodide assay was performed. The vital staining of CHO AA8 cells with acridine orange was applied to detect the development of acidic vesicular organelles (AVOs).Results: The performed experiments revealed a dose-dependent decrease in the cell survival. The morphological and ultrastructural features acquired by the cells after ATO-treatment were considered as typical for autophagy and mitotic cell death. As was shown by acridine orange staining, arsenic trioxide treatment increased red fluorescence signals in dose-dependent manner, indicating the development of AVOs, a hallmark of autophagy. Low level of apoptosis was induced in the ATO-treated CHO AA8 cells. Furthermore, the rearrangement of actin filaments associated with cell death process was also detected.Conclusions: The obtained results suggest that arsenic trioxide preferentially induces nonapoptotic cell deaths, autophagy and mitotic cell death, in p53-deficient CHO AA8 cells. Furthermore, the distinctive patterns of F-actin remodeling after As2O3 treatment were associated with different modes of cell death, confirming that cytoskeleton is a dynamic structure actively involved in the cell death process.

Introduction

Arsenic trioxide (ATO, As2 O3 ) is the chemotherapy agent approved by FDA for the treatment of relapsed or refractory acute promyelocytic leukemia (APL). Furthermore, it is also in clinical trials of other hematologic and solid tumors [20,57]. The extensive research demonstrating that ATO acts through the variety of intracellular signal transduction pathways to result in the inhibition of growth, the induction of cell death and the promotion of differentiation. However, the precise molecular mechanisms underlying the antitumor activity of As2O3 are still fully unknown, but seem to depend on cell types, dosage, and the duration of exposure [40]. Many reports have shown that ATOinduces cell death mainly via apoptotic pathways [12,13,14,15,33]. However, it is becoming increasingly clear that ATO-treated cells can also die by nonapoptotic mechanisms, such as mitotic catastrophe, autophagy and necrosis [53,34,44]. ATO-induced apoptosis is associated with the activation of caspase cascade, the inhibition of NFκB, the induction of oxidative stress, and the disruption of mitochondrial membrane potential. Moreover, changes in the expression of bcl-2, the activation of JNK kinase, and the inhibition of telomerase may also contribute to the induction of apoptosis by arsenic trioxide [5,10,40].

Previous studies revealed that p53 mutations are involved in 50% of all human cancers and nearly all chemotherapeutic drugs kill cancer cells mainly by apoptosis, that usually depends upon intact p53 signaling [16,18,47]. Thus, it is also extremely important to explore the mechanisms of anticancer drugs action that lead to nonapoptotic cell death induction such as mitotic catastrophe, because this type of cell death is not related to p53 tumor suppressor gene activity [29,45,48]. Hence, the drugs which are able to trigger the different types of cell death, offer a great potential for cancer therapy.

The biological responses to As2 O3 are likely to be partially caused by the modification of critical cysteine groups in cellular proteins [10,40]. Therefore, the proteins with a high cysteine content and accessible thiol groups are potent candidates for interactions with ATO. The cytoskeleton, that contains proteins with a relatively high sulfhydryl content is known to be a cellular target for arsenic trioxide [4,37,38,39]. Since various studies have implicated the actin cytoskeleton in both cell death and tumorigenesis, actin filaments constitute an attractive target for cancer therapy [17]. In the current study, the responses of CHO AA8 cells to arsenic trioxide were evaluated by characterizing cell viability, mechanisms of cell death and F-actin rearrangement. The choice of p53-lack cell line in present investigation in the manner of determination of cell death type seems justified.

MaterialsAndMethods

Cell culture and treatment

The Chinese hamster ovary cell line (CHO AA8) was kindly provided by Prof. M. Zdzienicka (Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, Poland). CHO AA8 cells were grown at 37°C in a humidified 5% CO2 atmosphere in minimum essential medium eagle (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Gibco) and the mixture of penicillin and streptomycin (Sigma-Aldrich) in concentration of 1ml/100ml medium. After 24 h of the culture, the cells were incubated with arsenic trioxide. ATO was added at doses of 1 μg/ml, 2 μg/ml and 3 μg/ml for 24 h. Control cells were cultured identically without ATO treatment. Cell viability was assessed by the trypan blue dye exclusion method.

Light microscopy

For the morphological analysis, the CHO AA8 cells grown on coverslips were fixed in 4% paraformaldehyde and then incubated with 0,1M glycine solution. The cells were stained with Mayer’s hematoxylin and rinsed under running tap water and dehydrated in a graded series of alcohols and xylenes. The preparations were observed using an Eclipse E800 microscope (Nikon) with NIS-Elements image analysis system and CCD camera (DS-5Mc-U1; Nikon).

Transmission electron microscopy

For the ultrastructural analysis, the CHO AA8 cells were fixed with 3,6% glutaraldehyde and moved to 0.1M cacodylate buffer (pH 7,4). Afterwards, the cells were postfixed with 1% osmium tetroxide, dehydrated with an ascending series of alcohols and acetones, and embedded in Epon 812. The polymerization of the resin occurred at 37°C for 24 h, and then at 65°C for 120 h. Selected parts of material were cut into ultra-thin sections by using Reichert OmU3 ultramicrotome and then counterstained with uranyl acetate and lead citrate. The material was examined using JEM 100 CX electron microscope (JEOL).

Image-Based Cytometer

For quantitative measurement of cell death, Tali® Apoptosis Assay Kit with Annexin V-Alexa Fluor® 488 and Propidium Iodide (Invitrogen) was used according to the manufacturer’s instructions. Briefly, the CHO AA8 cells were harvested from 6-well plates, centrifuged and the cell pellets were resuspended in 100 μl of Annexin Binding Buffer and 5 μl Annexin V-Alexa Fluor® 488. After 20 min incubation at room temperature in the dark, the cells were centrifuged at 300g for 5 min. The supernatants were discarded and the cell pellets were resuspended in 100 μl of Annexin Binding Buffer and 1 μl of propidium iodide. The incubation lasted for 5 min at room temperature in the dark. Preparated cells were analyzed with TaliTM-Image Based Cytometer. The double labeling procedure allows to distinguish early apoptotic (Annexin V – Alexa Fluor® 488+ /PI- ) from late apoptotic (Annexin V – Alexa Fluor® 488+ /PI+ ) and necrotic cells (Annexin V – Alexa Fluor® 488- /PI+ ).

Fluorescence microscopy

For the immunofluorescence labeling of F-actin, the CHO AA8 cells were fixed with 4% paraformaldehyde, embedded in 0.1 M glycine solution and thereafter permeabilized with 0.1% Triton X-100. The cells were incubated with phalloidin conjugated to Alexa Fluor 488 (Invitrogen, diluted 1:40) to enable the visualization of F-actin. The nuclei of the cells were labeled with 4’,6-diamidino- 2-phenyloindole (DAPI; Sigma-Aldrich, St. Louis, Missouri, USA). Slides were mounted in Aqua-Poly/Mount (Polysciences) and analysed by using an Eclipse E800 microscope with a Y-FL fluorescence attachment (Nikon), NIS-Elements 3.30 image analysis system and CCD camera (DS-5Mc-U1; Nikon).

Supravital cell staining with Acridine Orange (AO)

To detect the development of acidic vesicular organelles (AVOs), the vital staining of CHO AA8 cells with acridine orange was performed. Treated cells were stained with medium containing 1 μg/ml AO for 15 min at 37ºC, then washed twice in PBS and examined immediately with an Eclipse E800 microscope with a Y-FL fluorescence attachment (Nikon), NIS-Elements 3.30 image analysis system and CCD camera (DS-5Mc-U1; Nikon). The cytoplasm and nucleus of AO-stained cells fluoresced bright green, whereas the acidic autophagic vacuoles fluoresced bright red as described previously [51,54]. Fluorescence intensity was measured with ImageJ software (NIH).

Statistical analysis

GraphPad Prism 5.0 (GraphPad Software) was used for the statistical analysis. In order to assess the statistically significant differences between control and ATO doses, the nonparametric Mann-Whitney U test was performed. Results were considered at p<0.05.

Results

The viability of CHO AA8 cells after treatment with arsenic trioxide

The CHO AA8 cells were incubated with ATO for 24 h at concentrations 1, 2 and 3 μg/ml. The impact of ATO on cell viability was quantitated by the trypan blue dye exclusion assay. Figure 1 shows that the mean percentage of viable cells decreases significantly with dose of arsenic trioxide. Statistical analysis showed statistically significant differences (p<0.05) in the average percentage of surviving cells in comparison to control.

Qualitative and quantitative determinations of ATO-induced cell death in the CHO AA8 cells

Numerous reports have shown that ATO induces cell death in various cell lines, mainly through apoptotic pathways [12,13,14,15,33]. We therefore tested whether ATO triggers apoptosis in CHO AA8 cells. The light and transmission electron microscopy revealed little evidence of apoptosis in ATO-treated cells (Fig. 2,3). Only a few cells displayed typical morphologic changes associated with apoptosis like cell shrinkage and chromatin condensation (Fig. 2). To determine the proportion of cell death due to apoptosis, Annexin V-Alexa Fluor® 488 and Propidium Iodide assay was used following treatment with ATO. Annexin V is a calcium-dependent phospholipid binding protein that can be used to identify the externalization of phosphatidylserine during the early stage of apoptosis [60]. Propidium iodide (PI) is a DNA intercalating agent that may be incorporated into cells only after cell membrane damages [41]. In negative-control cells, the median viability rate was 94.45%, the median early apoptosis rate was 0.37% and the median late apoptosis rate was 2.95% (Fig. 5B,C,D,E). After the treatment with ATO at concentrations of 1, 2 and 3 μg/ml, there was a small but statistically significant increase in the percentages of early apoptotic cells (Fig. 5C). Similary, the percentage of late apoptotic cells increased slightly but significantly after treatment with ATO at 2 and 3 μg/ml, except for 1 μg/ml, in comparison to control (Fig. 5D). There was no significant difference in the percentage of necrotic cells between 1, 2, 3 μg/ml ATO and control (Fig. 5E). Likewise, the ATO exposed cells did not exhibit the morphologic hallmarks of necrosis (Fig. 2). These results indicate that apoptosis was not the main mode of cell death triggered in CHO AA8 cells exposed to ATO, and that necrosis was not induced in these cells following As2 O3 treatment.

Since recent studies have revealed that cancer cells undergo autophagy in response to various anticancer therapies [36], therefore we examined whether ATO induces autophagy in CHO AA8 cells. Autophagy is characterized by the formation of AVOs (acidic vesicular organelles), which include autophagic vacuoles and lysosomes [34]. The activation of autophagy was determined with transmission electron (TEM) microscopy and acridine orange (AO) labeling. In contrast to control cells, numerous AVOs were observed in the cytoplasm of ATO-treated CHO AA8 cells. The autophagic vacuoles contained cellular material that was at various stages of degradation (Fig. 3). To confirm the occurrence of autophagy in CHO AA8 cells, the AVOs were visualized by fluorescence microscopy following staining with acridine orange. AO is a fluorescent weak base that accumulates in acidic spaces, such as AVOs, and emits bright red fluorescence. The intensity of the red fluorescence is proportional to the degree of the acidity and volume of acidic vesicular organelles [43]. As shown in Fig. 4, arsenic trioxide treatment increased red fluorescence signals in dose-dependent manner, indicating the development of AVOs. Taken together, our observations suggest that ATO triggers autophagic death in the CHO AA8 cells.

A distinct morphology of CHO AA8 cells exposed to As2 O3 was the formation of giant cells with multiple nuclei of different sizes or one big nucleus (Fig. 2). The giant cells were several times bigger in size, compared to control cells. This phenotype appears to be consistent with mitotic cell death, as described previously [11,19].

Actin cytoskeleton rearrangement after treatment of CHO AA8 cells with ATO

Actin cytoskeleton is a dynamic structure actively involved in the cell death process [61]. In order to assess the reorganization of filamentous actin during cell death, the fluorescence microscopy analysis was performed following staining with phalloidin-Alexa Fluor® 488. In CHO AA8 control cells, F-actin was organized in stress fibers which traversed the cell. Labeling for F-actin was more pronounced at the cell cortex (Fig. 6A). There were no morphological changes in nuclei of control cells (Fig. 6B). After the treatment with ATO, many of CHO AA8 cells became detached and finally round up. In rounded cells, stress fibers were absent and F-actin accumulated beneath the plasma membrane to form cortical ring (Fig. 6C,E,G). The nuclei of most rounded cells did not exhibit other apoptotic features, such as fragmentation, chromatin condensation and margination as well (Fig. 6D,F,H). However, a high concentration of F-actin as aggregates or ring-like structures at the periphery of shrunken cells was occasionally observed (Fig. 6E,G). Furthermore, a few shrunken cells with condensed chromatin and depolymerization of actin filaments were also found, especially at the highest dose of ATO (Fig. 6G). Additionally, ATO treatment resulted in the appearance of numerous flattened giant cells with multiple nuclei (Fig. 6D,F,H,J) and strongly expanded actin network. In these cells, F-actin was visualized not only in stress fibers (Fig. 6C,E,G) but also in ruffled cell edges (Fig. 6C) and as aggregates in the cytoplasm (Fig. 6G).

Discussion

The anticancer drug arsenic trioxide is known to be an effective inducer of cell death in various cancer cell lines [40]. As it has been shown in numerous studies, the cytotoxic effect of ATO is mainly mediated through apoptosis [12,13,14,15,33]. Shim et al. observed apoptosis induction in chronic myelogenous leukemia K562 cells following arsenic trioxide treatment. They revealed the apoptotic morphological changes, characteristic pattern of nucleosomal DNA fragmentation and caspases activation in examined cells [50]. Recently, we have also found the hallmarks of apoptosis in K-562 cells exposed to As2 O3 [31]. Furthermore, we observed proapoptotic and antiproliferative effect of ATO in promyelocytic leukemia HL60 cells [32]. Similar results in HL-60 cells were also presented by Charoensuk et al. [13]. Besides, the occurrence of apoptosis in various solid tumors cell lines after As2 O3 treatment was detected as well. Wang et al. showed typical apoptotic morphology (such as condensed nuclei, membrane blebbing and the formation of apoptotic bodies) in human breast cancer MCF-7 cells incubated with As2 O3 [56]. Comparable findings were observed in esophageal carcinoma [49], neuroblastoma [1], prostate and ovarian carcinoma cells [55]. Based on the above results, we speculated that ATO may induced apoptosis in CHO AA8 cell line. Microscopic cellular analysis is considered as a key discriminant of different cell death types [6], thus we performed light, electron and fluorescence microscopy analysis of ATO-treated CHO AA8 cells. In addition, Annexin V-Alexa Fluor® 488/PI assay for estimating apoptotic cells was performed. Low level of apoptosis was induced in the ATO-treated CHO AA8 cells. Similarly, only a small population of ATO-exposed cells exhibited morphological features of apoptosis, including cytoplasmic and nuclear shrinkage as well as chromatin condensation. These results indicate that apoptosis was not the main type of cell death triggered in CHO AA8 cells incubated with ATO. In our previous studies using CHO AA8 cells, we observed apoptosis induction after treatment with ATO at concentrations 0.6 and 1.2 μg/ml. However, in those studies we did not perform quantitative evaluation of apoptosis, but we focused merely on the qualitative results [35].

Recent studies have shown that tumor cell response to chemotherapy is not confined to apoptosis but also includes nonapoptotic mechanisms, such as autophagy, mitotic catastrophe (mitotic cell death) and necrosis [16,47]. Since arsenic trioxide was shown to be able to induce autophagy [6,34,46], we looked for signs of autophagy in ATO-treated CHO AA8 cells. Autophagy is characterized by the formation of AVOs (acidic vesicular organelles), which include autophagic vacuoles and lysosomes. The development of AVOs is visualized and measured by vital staining of acridine orange [34,43]. In our studies, the transmission electron microscopy was used to examine the occurrence of autophagy in the ATO-treated CHO AA8 cells, because TEM is considered the most convincing method for analysis of autophagy [3]. The large amount of AVOs were found in the cytoplasm of CHO AA8 cells exposed to As2 O3 . We observed a lot of empty vacuoles as well as vacuoles in different degrees of degradation of their content. Moreover, the performed AO-staining confirmed that As2 O3 induced AVOs formation in CHO AA8 cells and revealed that this effect occurred in dose-dependent manner. The obtained results suggest that arsenic trioxide triggers autophagy in the CHO AA8 cells. Similar findings were presented by Kanzawa et al., who showed that the low concentration of ATO (2 μM) induced autophagic cell death, but not apoptosis in malignant glioma cell lines. Interestingly, they have also revealed that after exposure to low doses of ATO and bafilomycin A1 (an autophagy inhibitor), autophagy was inhibited and apoptosis occurred alternatively [34]. Furthermore, Bolt et al. suggest that in human lymphoblastoid cells autophagy is a key component in the cytotoxicity resulting from arsenite exposure. They observed autophagic vacuoles formation, acidic vesicle fluorescence, and LC3 expression (an autophagosome marker) in examined cells treated with sodium arsenite at concentration of 6 μM. On the other hand, markers indicative of apoptosis (phosphatidylserine externalization, PARP cleavage, and sensitivity to caspase) were negative in arsenite-treated cells [6].

Cell death by mitotic catastrophe (MC) has recently been proposed [11,19,48]. MC was defined by Castedo et al. as a type of cell death occurring during mitosis that results from cellular damage as well as the combination of deficient cell-cycle checkpoints. However, among the scientists there was no consensus in defining mitotic catastrophe and the molecular mechanisms leading to MC are still largely unclear. Morphologically, mitotic cell death is characterized by the formation of giant cells that contain nuclei with altered morphology, e.g. multiple nuclei or micronuclei [11]. Here, after the treatment of CHO AA8 cells with ATO, the appearance of numerous enlarged cells with one big nucleus or multiple nuclei of different sizes was seen. Recently, it has been proposed that arsenic compounds are mitotic disruptors causing mitotic abnormalities [52,53], which can lead to mitotic catastrophe as described previously [11,19]. In the study shown by Yih et al., arsenite triggered the centrosome amplification, spindle multipolarity and chromosome missegregation leading to mitotic arrest and mitotic cell death in CGL-2 cells [59]. The same authors have also revealed that sodium arsenite induced multinucleation, endoreduplication, aneuploidy and delayed normal mitotic progression in human fibroblasts [58]. Importantly, accumulating evidences indicate that MC occurs preferentially in p53 deficient cells [29,45,48], whereas apoptosis is usually associated with functional wild-type p53 activity [40]. Ianzini et al. demonstrated that MC was induced in human tumor cells such as HeLa [30] and PC-3 cells [28], which are both impaired on their p53 functionality. On the contrary, in the human glioblastoma U87MG cells with functional p53, MC was not observed [29]. Moreover, Taylor et al., using TR9-7 cells (a model cell line with p53 exogenously regulated in a tetracycline-off expression system), showed that arsenite treatment triggered the incidence of mitotic catastrophe to a greater extent in p53- negative than in p53-positive cells. They concluded that p53 expression plays a pivotal role in preventing mitotic catastrophe induced by arsenite [52]. Based on the above results, we presume that the lack of functional p53 in CHO AA8 cell line potentiates mitotic cell death induction by arsenic trioxide. This result suggests, that ATO might be a beneficial drug for the treatment p53 deficient cancers, and supports conclusions presented by others [18,27].

Additionally, in the current study the reorganization of actin filaments after treatment of CHO AA8 cells with ATO were observed. Numerous studies have shown that actin cytoskeleton is a dynamic structure actively involved in the realization of cell death process [23,25,61]. The rearrangement of F-actin accompaning apoptosis was well documented in many cell lines treated with various anticancer agents. Grzanka et al. suggested that there is a correlation between F-actin localization and apoptotic body formation during the apoptosis [23]. The same authors have also revealed the results where the F-actin was presented in the nucleus area during apoptosis and hypothesized that the function of nuclear F-actin may be involved in chromatin remodeling [24]. Besides, it has been documented that depolymerization or cleavage of microfilament networks and other cytoskeletal elements (e.g. microtubules and lamins) also is required for major morphological changes that occur with apoptosis [7,8]. Here, a high concentration of Factin as aggregates or ring-like structures at the periphery of shrunken cells was occasionally observed. Moreover, a few shrunken cells with condensed chromatin as well as depolymerization of actin cytoskeleton was also found, especially at the highest dose of ATO. Based on the abovecited studies, we suggest that observed actin cytoskeleton rearrangement is required for the main morphological changes associated with apoptosis.

To date, less pharmacological research has been devoted to the study of actin remodeling during autophagy. Bursch et al. pointed that autophagic and apoptotic types of cell death exhibit different fates of cytoskeletal filaments. These authors used tryphostin A25-treated human colon cancer cells (HT29/HI1) as a model of apoptosis, whereas autophagy was induced by its known inducer, tamoxifen, in human breast cancer cell line (MCF-7). In early apoptotic cells, they observed actin depolymerization and degradation of intermediate filaments. On the contrary, during autophagic cell death actin and intermediate filaments were redistributed, but largely preserved even beyond the stage of nuclear collapse. In autophagic cells, actin was found to be polymerized to filaments, which aggregated around the nucleus with star-like extension to the cell membrane [9]. Here, as it has been shown by phalloidin staining, the actin was still present in the form of filaments after the treatment of CHO AA8 cells with arsenic trioxide. We observed actin cytoskeleton rearrangement, but in the depolimerized state, actin was found only in the few cells exhibiting the morphological features of apoptosis. These observations are consistent with those presented by Bursch et al [9]. The intact cytoskeleton has previously been shown to be necessary during autophagy. Aplin et al. revealed that intermediate and microfilaments are involved in the initial formation of autophagosomes [2]. Likewise, microtubule was demonstrated to be required for fusion between autophagosomes and lysosomes to form autolysosomes [26,21], the degradative autophagic structures containing partially degraded cellular material [3].

In present study, we also noted strongly expanded actin network in cells with the phenotype resembling mitotic cell death. In these cells, F-actin was visualized not only in stress fibers but also in ruffled cell edges and as aggregates in the cytoplasm. Grzanka et al. have also reported the appearance of giant multinucleated cells with extended network of fine microfilaments after exposure of CHO AA8 cells to doxorubicin [25]. Similar results were described by Nowak et al., in the non-small-cell lung cancer cells (A549) incubated with cotinine [42]. Likewise, Gonçalves et al. observed overexpression of the phalloidin stainingactin filaments in thyroid papillary carcinoma cells (TPC- 1) undergoing mitotic catastrophe under the influence of rotenone [22].

In conclusion, our results showed that ATO reduced viability of CHO AA8 cells in dose dependent manner. These data also suggest that arsenic trioxide preferentially induces nonapoptotic cell deaths, autophagy and mitotic cell death, in p53-deficient CHO AA8 cells. Furthermore, the distinctive patterns of F-actin remodeling after As2 O3 treatment were associated with different modes of cell death, confirming that cytoskeleton is a dynamic structure actively involved in the cell death process.

References

• 1. Akao Y., Nakagawa Y., Akiyama K.: Arsenic trioxide induces apoptosisin neuroblastoma cell lines through the activation of caspase 3 in vitro.FEBS Lett., 1999; 455: 59-62
  Google Scholar
• 2. Aplin A., Jasionowski T., Tuttle D.L., Lenk S., Dunn W.A.Jr.: Cytoskeletalelements are required for the formation and maturation of autophagicvacuoles. J. Cell Physiol., 1992; 153: 458-466
  Google Scholar
• 3. Barth S., Glick D., Macleod K.F.: Autophagy: assays and artifacts. JPathol., 2010; 221: 117-124
  Google Scholar
• 4. Binet F., Cavalli H., Moisan E., Girard D.: Arsenic trioxide (AT) is anovel human neutrophil pro-apoptotic agent: effects of catalase onAT-induced apoptosis, degradation cytoskeletal proteins and de novoprotein synthesis. Br. J. Haematol., 2006; 132: 349-358
  Google Scholar
• 5. Bode A.M., Dong Z.: The paradox of arsenic: molecular mechanismsof cell transformation and chemotherapeutic effects. Crit. Rev. Oncol.Hematol., 2002; 42: 5-24
  Google Scholar
• 6. Bolt A.M., Byrd R.M., Klimecki W.T.: Autophagy is the predominantprocess induced by arsenite in human lymphoblastoid cell lines. Toxicol.Appl. Pharmacol., 2010; 244: 366-373
  Google Scholar
• 7. Bonfoco E., Leist M., Zhivotovsky B., Orrenius S., Lipton S.A., NicoteraP.: Cytoskeletal breakdown and apoptosis elicited by NO donors incerebellar granule cells require NMDA receptor activation. J. Neurochem.,1996; 67: 2484-2493
  Google Scholar
• 8. Brown S.B., Bailley K., Savill J.: Actin is cleaved during constitutiveapoptosis. Biochem. J., 1996; 323: 233-237
  Google Scholar
• 9. Bursch W., Hochegger K., Török L., Marian B., Ellinger A., HermannR.S.: Autophagic and apoptotic types of programmed cell death exhibitdifferent fates of cytoskeletal filaments. J. Cell Sci., 2000; 113:1189-1198
  Google Scholar
• 10. Carney D.A.: Arsenic trioxide mechanisms of action – looking beyondacute promyelocytic leukemia. Leuk. Lymphoma, 2008; 49: 1846-1851
  Google Scholar
• 11. Castedo M., Perfettini J.L., Roumier T., Andreau K., Medema R.,Kroemer G.: Cell death by mitotic catastrophe: a molecular definition.Oncogene, 2004; 23: 2825-2837
  Google Scholar
• 12. Cha Y., Park D.W., Lee C.H., Baek S.H., Kim S.Y., Kim J.R., Kim J.H.:Arsenic trioxide induces apoptosis in human colorectal adenocarcinomaHT-29 cells through ROS. Cancer Res. Treat., 2006; 38: 54-60
  Google Scholar
• 13. Charoensuk V., Gati W.P., Weinfeld M., Le X.C.: Differential cytotoxiceffects of arsenic compounds in human acute promyelocytic leukemiacells. Toxicol. Appl. Pharmacol., 2009; 239: 64-70
  Google Scholar
• 14. Chen G.Q., Zhu J., Shi X.G., Ni J.H., Zhong H.J., Si G.Y., Jin X.L., TangW., Li X.S., Xong S.M., Shen Z.X., Sun G.L., Ma J., Zhang P., Zhang T.D.,Gazin C., Naoe T., Chen S.J., Wang Z.Y., Chen Z.: In vitro studies on cellularand molecular mechanisms of arsenic trioxide (As2O3) in the treatmentof acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosiswithdown regulation of Bcl-2 expression and modulation of PML-RAR α/PML proteins. Blood, 1996; 88: 1052-1061
  Google Scholar
• 15. Chen M.J., Yang P.Y., Ye Y.Z., Hu D.N., Chen M.F.: Arsenic trioxideinduces apoptosis in uveal melanoma cells through the mitochondrialpathway. Am J. Chin. Med., 2010; 38: 1131-1142
  Google Scholar
• 16. De Bruin E.C., Medema J.P.: Apoptosis and nonapoptotic deathsin cancer development and treatment response. Cancer Treat. Rev.,2008; 34: 737-749
  Google Scholar
• 17. Desouza M., Gunning P.W., Stehn J.R.: The actin cytoskeleton as asensor and mediator of apoptosis. BioArchitecture, 2012; 2: 75-87
  Google Scholar
• 18. Dong Z.: The molecular mechanisms of arsenic-induced cell transformationand apoptosis. Environ. Health Perspect., 2002; 110, Suppl.5: 757-759
  Google Scholar
• 19. Erenpreisa J., Cragg M.S.: Mitotic death: a mechanism of survival?A review. Cancer Cell Int., 2001; 1: 1
  Google Scholar
• 20. Evens A.M., Tallman M.S., Gartenhaus R.B.: The potential of arsenictrioxide in the treatment of malignant disease: past, present, and future.Leukemia Res., 2004; 28: 891-900
  Google Scholar
• 21. Fengsrud M., Roos N., Berg T., Liou W.L., Slot J.W., Seglen P.O.: Ultrastructuraland immunocytochemical characterization of autophagicvacuoles in isolated hepatocytes: effects of vinblastine and asparagineon vacuole distributions. Exp. Cell Res., 1995; 221: 504-519
  Google Scholar
• 22. Gonçalvesa A.P., Máximo V., Limac J., Singhe K.K., Soares P., VideiraA.: Involvement of p53 in cell death following cell cycle arrest and mitoticcatastrophe induced by rotenone. Biochim. Biophys. Acta, 2011;1813: 492-499
  Google Scholar
• 23. Grzanka A., GrzankaD., Orlikowska M.: Cytoskeletal reorganizationduring process of apoptosis induced by cytostatic drugs in K-562 andHL-60 leukemia cell lines. Biochem. Pharmacol., 2003; 66: 1611-1617
  Google Scholar
• 24. Grzanka A., Grzanka D., Orlikowska M.: Fluorescence and ultrastructurallocalization of actin distribution patterns in the nucleusof HL-60 and K-562 cell lines treated with cytostatic drugs. Oncol.Rep., 2004; 11: 765-770
  Google Scholar
• 25. Grzanka D., Grzanka A., Izdebska M., Gackowska L., Stepien A.,Marszalek A.: Actin reorganization in CHO AA8 cells undergoingmitotic catastrophe and apoptosis induced by doxorubicin. OncolRep., 2010; 23: 655-663
  Google Scholar
• 26. Høyvik H., Gordon P.B., Berg T.O., Strømhaug P.E., Seglen P.O.:Inhibition of autophagiclysosomal delivery and autophagic lactolysisby asparagine. J. Cell Biol., 1991; 113: 1305-1312
  Google Scholar
• 27. Huang C., Ma W.Y., Li J., Dong Z.: Arsenic induces apoptosisthrough a c-Jun NH2-terminal kinase-dependent, p53-independentpathway. Cancer Res., 1999; 59: 3053-3058
  Google Scholar
• 28. Ianzini F., Bertoldo A., Gesie A., Kosmacek E.A., Nguyen T., MackeyM.A.: Adriamycin induces mitotic catastrophe in PC-3 humanprostate cancer cells. 51st Annual Meeting of the Radiation ResearchSociety. St. Louis, MO, April 24-27, 2004
  Google Scholar
• 29. Ianzini F., Bertoldo A., Kosmacek E.A., Phillips S.T., Mackey M.A.:Lack of p53 function promotes radiation-induced mitotic catastrophein mouse embryonic fibroblast cells. Cancer Cell Int., 2006; 6: 11
  Google Scholar
• 30. Ianzini F., Mackey M.A.: Spontaneous premature chromosomecondensation and mitotic catastrophe following irradiation of HeLaS3 cells. Intl. J. Radiat. Biol., 1997; 72: 409-421
  Google Scholar
• 31. Izdebska M., Grzanka A., Ostrowski M., Żuryń A., Grzanka D.:Effect of arsenic trioxide (Trisenox) on actin organization in K-562erythroleukemia cells. Folia Histochem. Cytobiol., 2009; 47: 453-459
  Google Scholar
• 32. Izdebska M., Grzanka D., Gackowska L., Żuryń A., Grzanka A.:The influence of Trisenox on actin organization in HL-60 cells. Cent.Eur. J. Biol., 2009; 4: 351-361
  Google Scholar
• 33. Jing Y., Dai J., Chalmers-Redman R.M., Tatton W.G., Waxman S.:Arsenic trioxide selectively induces acute promyelocytic leukemiacell apoptosis via a hydrogen peroxide-dependent pathway. Blood,1999; 94: 2102-2111
  Google Scholar
• 34. Kanzawa T., Kondo Y., Ito H., Kondo S., Germano I.: Induction ofautophagic cell death in malignant glioma cells by arsenic trioxide.Cancer Res., 2003; 63: 2103-2108
  Google Scholar
• 35. Klimaszewska A., Izdebska M., Gagat M., Grzanka D., GrzankaA.: The influence of arsenic trioxide on the reorganization of thetubulin protein in CHO AA8 cell line. Med. Biol. Sci., 2011; 25: 17-23
  Google Scholar
• 36. Kondo Y., Kanzawa T., Sawaya R., Kondo S.: The role of autophagyin cancer development and response to therapy. Nat. Rev. Cancer,2005; 5: 726-734
  Google Scholar
• 37. Li W., Chou I.N.: Effects of sodium arsenite on the cytoskeletonand cellular glutathione levels in cultured cells. Toxicol. Appl. Pharmacol.,1992; 114: 132-139
  Google Scholar
• 38. Li Y.M., Broome J.D.: Arsenic targets tubulins to induce apoptosisin myeloid leukemia cells. Cancer Res., 1999; 59: 776-780
  Google Scholar
• 39. Menzel D.B., Hamadeh H.K., Lee E., Meacher D.M., Said V., RasmussenR.E., Greene H., Roth R.N.: Arsenic binding proteins fromhuman lymphoblastoid cells. Toxicol. Lett., 1999; 105: 89-101
  Google Scholar
• 40. Miller W.H., Hyman M., Schiooer M., Lee J.S., Singer J., WaxmanS.: Mechanism of action of arsenic trioxide. Cancer Res., 2002;62: 3893-3903
  Google Scholar
• 41. Nicoletti I., Migliorati G., Pagliacci M.C., Grignani F., Riccardi C.:A rapid and simple method for measuring thymocyte apoptosis bypropidium iodide staining and flow cytometer. J. Immunol. Methods,1991; 139: 271-276
  Google Scholar
• 42. Nowak J.M., Grzanka A., Gagat M., Żuryń A.: The influence ofcotinine on the non-small-cell lung cancer line A549. Postępy Hig.Med. Dośw., 2009; 63: 1-7
  Google Scholar
• 43. Paglin S., Hollister T., Delohery T., Hackett N., McMahill M.,Sphicas E., Domingo D., Yahalom J.: A novel response of cancer cellsto radiation involves autophagy and formation of acidic vesicles.Cancer Res., 2001; 61: 439-444
  Google Scholar
• 44. Pettersson H.M., Pietras A., Munksgaard Persson M., KarlssonJ., Johansson L., Shoshan M.C. Påhlman S.: Arsenic trioxide is highlycytotoxic to small cell lung carcinoma cells. Mol. Cancer Ther.,2009; 8: 160-170
  Google Scholar
• 45. Portugal J., Mansilla S., Bataller M.: Mechanisms of drug-inducedmitotic catastrophe in cancer cells. Curr. Pharm. Des., 2010; 16: 69-78
  Google Scholar
• 46. Qian W., Liu J., Jin J., Ni W., Xu W.: Arsenic trioxide induces notonly apoptosis but also autophagic cell death in leukemia cell linesvia up-regulation of Beclin-1. Leuk. Res., 2007; 31: 329-339
  Google Scholar
• 47. Ricci M.S., Zong W.X.: Chemotherapeutic approaches for targetingcell death pathways. The Oncologist, 2006; 11: 342-357
  Google Scholar
• 48. Roninson I.B., Broude E.V., Chang B.D.: If not apoptosis, thenwhat? Treatment-induced senescence and mitotic catastrophe intumor cells. Drug Resist. Update, 2001; 4: 303-313
  Google Scholar
• 49. Shen Z.Y., Shen J., Cai W.J., Hong C., Zheng M.H.: The alterationof mitochondria is an early event of arsenic trioxide-induced apoptosisin esophageal carcinoma cells. Int. J. Mol. Med., 2000; 5: 155-158
  Google Scholar
• 50. Shim M.J., Kim H.J., Yang S.J., Lee I.S., Choi H.I., Kim T.: Arsenictrioxide induces apoptosis in chronic myelogenous leukemia K562cells: possible involvement of p38 MAP kinase. Biochem. Mol. Biol.,2002; 35: 377-383
  Google Scholar
• 51. Stankiewicz M., Jonas W., Hadas E., Cabaj W., Douch P.G.: Supravitalstaining of eosinophils. Int. J. Parasitol., 1996; 26: 445-446
  Google Scholar
• 52. Taylor B.F., McNeely S.C., Miller H.L., Lehmann G.M., McCabeM.J.Jr., States J.C.: p53 suppression of arsenite-induced mitoticcatastrophe is mediated by p21CIP1/WAF1. J. Pharmacol. Exp. Ther.,2006; 318: 142-151
  Google Scholar
• 53. Taylor B.F., McNeely S.C., Miller H.L., States J.C.: Arsenite-inducedmitotic death involves stress response and is independent oftubulin polymerization. Toxicol. Appl. Pharmacol., 2008; 230: 235-246
  Google Scholar
• 54. Traganos F., Darzynkiewicz Z.: Lysosomal proton pump activity:supravital cell staining with acridine orange differentiates leukocytesubpopulations. Methods Cell Biol., 1994; 41: 185-194
  Google Scholar
• 55. Uslu R., Sanli U.A., Sezgin C., Karabulut B., Terzioglu E., OmayS.B., Goker E.: Arsenic trioxide-mediated cytotoxicity and apoptosisin prostate and ovarian carcinoma cell lines. Clin. Cancer Res.,2000; 6: 4957-4964
  Google Scholar
• 56. Wang Y., Zhang Y., Yang L., Cai B., Li J., Zhou Y., Yin L., Yang L.,Yang B.F., Lu Y.: Arsenic trioxide induces the apoptosis of human breastcancer MCF-7 cells through activation of caspase-3 and inhibitionof HERG channels. Exp. Ther. Med., 2011; 2: 481-486
  Google Scholar
• 57. Waxman S., Anderson K.C.: History of the development of arsenicderivatives in cancer therapy. Oncologist, 2001; 6: 3-10
  Google Scholar
• 58. Yih L.H., Ho I.C., Lee T.C.: Sodium arsenite disturbs mitosis andinduces chromosome loss in human fibroblasts. Cancer Res., 1997;57: 5051-5059
  Google Scholar
• 59. Yih L.H., Tseng Y.Y., Wu Y.C., Lee T.C.: Induction of centrosomeamplification during arsenite-induced mitotic arrest in CGL-2 cells.Cancer Res., 2006; 66: 2098-2106
  Google Scholar
• 60. Zhang G., Gurtu V., Kain S.R. Yan G.: Early detection of apoptosisusing a fluorescent conjugate of annexin V. BioTechniques,1997; 23: 525-531
  Google Scholar
• 61. Zitterbart K., Veselská R.: Effect of retinoic acid on the actin cytoskeletonin HL-60 cells. Neoplasma, 2001; 48: 456-461
  Google Scholar

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