Abstract
Background/Aim: Antimony is a chemical element used in the therapy of parasitic diseases with a promising anticancer potential. The aim of this study was to evaluate in vitro activity of free or liposomal vesicle-packed antimony trioxide (AT or LAT) in the t(15;17)(q22;q21) translocation-positive acute promyelocytic leukemia (APL) cell line NB4. Materials and Methods: Cytotoxicity was analysed with trypan blue exclusion, the MTT assay and neutral red exclusion assay; cell proliferation with PicoGreen®; and reactive oxygen species (ROS) production with DCFDA. Results: Liposomal particles did not change the pH of the cell culture medium and entered the cells. Both formulations resulted in a time- and concentration-dependent cytotoxicity and production of ROS. LAT showed higher toxicity at lower concentrations compared to AT. Conclusion: LAT may be used to decrease drug dosage and maintain high anti-tumoral effects on APL cells.
Cancer is a multifactorial disease, and therefore its treatment needs to address different targets to achieve better results. New therapeutic strategies are investigated and repositioning of drugs is a promising alternative. Antimony is used in the therapy of leishmaniasis and schistosomiasis. The main serious adverse effects are associated to cardiotoxicity in about 9% of the patients and pancreatitis, more commonly seen in co-infections (HIV and visceral leishmaniasis) (1). Reactive oxygen species (ROS) production and oxidative stress damage are the main mechanisms of cell damage caused by antimony (2). Studies have shown that autophagy induced by antimony is triggered by the production of ROS and not by the activation of the mTOR pathway (3). In the present study, we used liposomes, which have low biological impact (4). Nanoparticles can improve drug delivery and efficiency at a lower dosage, reducing toxic effects, which is highly needed in cancer treatment (5). Therefore, we compared the effects of free antimony trioxide (AT) and AT encapsulated in liposomes (LAT) on the acute promyelocytic leukemia (APL) cell line NB4.
APL is caused by the fusion protein PML/RARA and was the first leukemia treated with the specific molecular targeting drug all-trans-retinoic acid (ATRA) (6). Despite the high efficiency of the treatment, 5 to 20% of the patients develop the ATRA syndrome, a life-threatening condition associated with organ infiltration by myeloid cells, cytokine production, high expression of CD13 in leukemic cells, and hyperleukocytosis, leading to endothelial inflammation and bleeding in the affected organs (6). Arsenic Trioxide (ATO) is an efficient drug for APL treatment, and in association with ATRA, decreases relapse in about 90% of cases (7), but its use is limited by the risk of toxicity and potential carcinogenicity (8).
The free antimony trioxide (Sb2O3) shows chemical structure and mechanistic properties similar to ATO against APL. They both induce apoptosis in a dose- and time-dependent manner. In addition, both compounds are able to induce degradation of the PML/RARA fusion protein (9). However, the application of antimony has clinical limitations due to the toxic effects associated with the metabolic products deposited in untargeted organs as well as to its hydrophobic characteristic. The use of drugs encapsulated in liposomes is preferred in clinical applications, compared to free drugs, as they show increased vascular permeability (<200 nm in diameter) and increased accumulation in targeted organs (10).
In this study, we used antimony as an alternative anticancer agent for the treatment of APL relapse. We evaluated the efficacy of LAT compared to free AT in APL cells. The choice for the use of liposomes was due to their small size and large surface area that add specific properties, including the ability to catalyse chemical reactions. The increased surface reactivity predicts greater biological activity compared to larger particles when absorbed by living organisms (11). We examined the effects of AT and LAT on the viability of the APL cell line NB4 and ROS production.
Materials and Methods
Production of liposomes containing antimony trioxide. Liposomes containing AT were obtained through the reverse-phase evaporation method (12). The physico-chemical assessments of the obtained formulation indicated a pH=7.28, size=80.05 nm, polydispersity index=0.279, Zeta potential=(–) 6.81, and Sb concentration 0.998±0.002 mg/ml. The encapsulation efficiency was 8.12±0.22% and the formulation remained stable for up to 30 days when stored at 4°C.
Cell culture and treatments. Cells were transferred into cell culture flasks containing RPMI 1640 (Sigma-Aldrich, São Paulo, Brazil), 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Sigma-Aldrich). Cell culture medium was changed every two to three days according to the American Type Culture Collection (ATCC) recommendations.
NB4 cells were plated into 96-well plates at the concentration of 1×104 cells/well and treated with 25, 50, 100, 200, 300, 400, or 500 μg/ml LAT, empty liposomes (EL) or free antimony trioxide (AT) at the same concentrations as the liposomal antimony. Plates were incubated for 24 and 72 h under the same conditions.
Trypan Blue exclusion test of cell viability. After incubation, the cells were transferred into 1.5 ml polypropylene tubes and centrifuged at 400 × g. Supernatant was discarded and cell pellet was gently suspended in RPMI 1640 medium, and 0.4% Trypan Blue (Sigma-Aldrich) was added to the cells (1:1). After 5 min at room temperature, the cells were transferred to Neubauer chamber and observed under the microscope. One hundred cells (non-stained and blue cells) were counted, and the percentage of non-stained cells represented viable cells.
Reduction of MTT reagent. After the incubation periods, 20 μl of the reagent (5 mg/mL) was added to each well containing cells staying in the CO2 incubator for 3 hours. Finally, the supernatant was removed and the product accumulated inside the cell was extracted by adding DMSO (Synth) (13). Treated cells were exposed to MTT and absorbance was measured at 570 nm in a microplate spectrophotometer (SpectraMax® i3x, Molecular Devices, San Jose, CA, USA).
Neutral red cytotoxicity assay. The supernatant was discarded, the pellet suspended in cell culture medium and 104 cells were plated into each well of a 96-well plate. The cells were treated, and after incubation, the medium was replaced with 200 μl of cell culture medium containing 0.04 μg/ml neutral red. The plates were incubated at 37°C and 5% CO2 for 4 h. The extraction of neutral red incorporated by the lysosomes of viable cells was performed with 200 μl solution of 1% acetic acid, 50% ethanol and 49% water. After extraction, the intensity of the colour was measured by spectrophotometry at 540 nm (14).
PicoGreen® fluorescence cytotoxicity test. PicoGreen® analysis was performed as previously described (15) using the fluorescent dye dsDNAPicoGreen® (Invitrogen, Paisley, UK). After the treatment and incubation of cells, as previously described, the cell culture plate was centrifuged and 10 μl of supernatant of each well were transferred into another 96-well plate. Eighty μl of tris-EDTA 1X buffer (Tris HCl 10 mmol/l, pH=7.5) and 5 μl of the PicoGreen® reagent were then added. The cells were incubated at room temperature for 5 min in the dark and the absorbance was measured by fluorimetry (SpectraMax® i3x – Molecular Devices) at 520 nm emission and 480 nm excitation. The results were expressed as fluorescence values indicating double strand DNA integrity.
Analysis of ROS production with 2’,7’-Dichlorofluorescein diacetate (DCF-DA). After treatment and incubation, the cells were transferred in the dark in 96-well plates and suspended in 50 μl cell culture medium containing 65 μl Tris HCl 10 mmol/l and 10 μl DCFH-DA. The plates were maintained for 30 min in a cell culture incubator (16). After incubation, fluorescence was measured using a spectrofluorometer (SpectraMax® i3x – Molecular Devices).
Statistical analysis. The results of cell viability were expressed as a percentage of negative control±standard deviation. The data were statistically analyzed by one-way (ANOVA) followed by Tukey post hoc using Graphpad prism® software (San Diego, CA, United States), version 5.0 (p-Values <0.05 were considered statistically significant).
Results
Effects of AT and LAT on cell viability - Trypan Blue exclusion cell viability test. After 24 h of incubation, treatment with 200, 300, 400 and 500 μg/ml AT reduced cell viability to 59±4%, 70±1%, 69±5% and 72±2%, respectively (p<0.001). Treatment with 200, 400 and 500 μg/ml LAT for 24 h reduced cell viability to 48±6%, 50±4% and 42±3%, respectively, (p<0.001) (Figure 1A). After 72 h incubation 300, 400 and 500 μg/ml of LAT reduced cell viability to 44±5%, 49±5% and 36±4%, respectively (Figure 1B) (p<0.001).
Cytoxicity and ROS production by free (AT) and liposomal vesiclepacked antimony trioxide (AT and LAT, respectively). (A) and (B) Trypan Blue assay; (C) and (D) MTT assay, (E) and (F) Neutral Red assay, (G) PicoGreen and (H) DCFH-DA assay. Treatments were compared to the negative control (untreated cells) *p<0.05, **p<0.01 and ***p<0.001. LAT was compared to AT #p<0.05, ##p<0.01 and ###p<0.001.
Considering toxicity versus concentration, we observed that AT at 400 and 500 μg/ml was more toxic than ATO in the same concentrations after 24 h. After 72 h, all concentrations, except for 50 μg/ml, showed better activity.
No changes in cell viability were observed following treatment with empty liposomes (EL) at all incubation times. Cell viability according to trypan blue analyses after 24- and 72-h of incubation is shown in Figure 1.
Effects of AT and LAT on cell viability – MTT assay. After 24 h, 25, 50, 100 and 300 μg/ml of LAT decreased cell viability compared to the same concentrations of AT (Figure 1C). After 72 h, both AT and LAT reduced cell viability to 15% at the lowest concentration (25 μg/ml). After 72 h incubation, treatment with 400 and 500 μg/ml EL reduced cell viability to 52±2% and 34±4%, respectively (Figure 1D).
Effects of AT and LAT on cell viability - neutral red assay. Using the method that considers the incorporation of neutral red in the lysosomes of viable cells, a higher viability was observed in cells treated with free AT compared to LAT. After 24 h of incubation, treatment with 500 μg/ml AT reduced cell viability to 56±3%, whereas approximately the same effect (50±6% reduction in cell viability) was obtained following treatment with 10 μg/ml of LAT (Figure 1E). After 72 h, AT toxicity increased (42±5% cell viability at 500 μg/ml) (p<0.001), whereas LAT toxicity at the same concentration was higher (26±3% cell viability) (Figure 1F). At the concentration of 25 μg/ml, AT was more toxic to the cells than LAT, and, at higher concentrations (100, 200, 300 μg/ml), LAT was more toxic after 24 h. LAT was also more toxic after 72 h at all concentrations.
PicoGreen® fluorescence cytotoxicity test. DNA damage following AT and LAT treatment for 72 h was assessed with the PicoGreen® assay. Treatment with 200 μg/ml AT increased extracellular dsDNA 100% in comparison to non-treated cells (p<0.01). LAT treatment was cytotoxic at 200 and 300 μg/ml concentrations (100 and 107%, respectively) (Figure 1G).
Analysis of ROS production with 2’,7’-Dichlorofluorescein diacetate (DCF-DA). In order to analyse ROS production in NB4 cells treated with AT and LAT, we chose the 72h incubation time. AT treatment increased ROS levels at 300, 400 and 500 μg/ml concentrations (p<0.001). LAT treatment induced ROS production at 200, 300, 400 and 500 μg/ml (p<0.001) and this production was the highest at 200 μg/ml (Figure 1H). LAT treatment induced higher ROS production in comparison to AT at 200, 300, and 500 μg/ml concentrations.
Discussion
Liposomal formulations are already used for cancer chemotherapy and, in the field of hematologic malignancies, liposomal vincristine (Marqibo) was approved by the FDA to treat acute lymphoblastic leukemia in 2012 and liposomal doxorubicin in the last years (17). A liposomal formulation of cytarabine and daunorubicin (CPX351), developed by Celator Pharmaceuticals Inc., showed promising results in a clinical phase III trial in patients with AML, with a response of 40% in induction (18). Particle size was defined to a maximum of 200 nm, that penetrate through naturally fenestrated endothelium in the bone marrow (19). The polydispersity index (PDI) <0.500 was defined as satisfactory for this experiment suggesting good homogeneity of the system (4). For these reasons, the size and characteristics of liposomes are important as they determine their transport through the blood stream and uptake into tumour cells (20). AT and LAT showed a dose-dependent cytotoxicity (Figure 1), which was higher after 72 h incubation. LAT showed cytotoxicity at lower concentrations in comparison to AT. This could be explained by a more efficient incorporation of the particle by the cells, in comparison to the free compound. Free compounds might also form insoluble complexes at higher concentrations, which reduces their biological activity.
Antimony complexes have also been shown to be toxic in HL-60 APL cells following treatment for 24 h. Similarly, antimony (III) has been shown to reduce viability of a murine leukemia cell line (L1210) (21). Taken together, our results are promising and offer a new therapeutic possibility to treat cancer with lower drug concentrations.
Liposomes obtained in our study were able to deliver efficiently AT to NB4 cells. A concentration- and time-dependent manner toxicity was observed and detected at lower concentrations in the liposomal formulation in comparison to free AT. LAT treatment induced ROS production in NB4 cells and provides a possible mechanism to efficiently treat APL with AT. In vivo studies are necessary to test the efficiency and safety of LAT, especially in comparison to the current therapy of APL.
Acknowledgements
This study was financed in part by the CAPES, Brazil – Finance Code 001.
Footnotes
Authors’ Contributions
All Authors contributed to the writing of the manuscript. All Authors reviewed and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare no potential conflicts of interest in relation to this study.
- Received August 25, 2021.
- Revision received September 15, 2021.
- Accepted September 21, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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