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. 2019 Jan 25;43(2):214–219. doi: 10.1002/cbin.11095

Multidrug resistance phenotype: Relation between phenotype induction and its characteristics in erythroleukemia cells

Fernanda Saldanha Soares 1,2, Aline Portantiolo Lettnin 1,2, Eduardo Felipe Wagner 2,3, Francielly Hafele Mattozo 2, Michele Carrett‐Dias 1,2, Vivian Mary Barral Dodd Rumjanek 4, Daza Moraes Vaz Batista Filgueira 2, Ana Paula de Souza Votto 1,2,✉
PMCID: PMC13397387  PMID: 30597722

Abstract

Chemotherapy may be followed by multiple drug resistance (MDR). This is an obstacle in the treatment of cancer. It is therefore essential to understand the mechanisms underlying tumor resistance, especially those involved in the cell target/MDR relationship. To investigate this, the effects of exposing cells to UVB (to target DNA), UVA, and H2O2 (to target the cell membrane) were observed in K562 (non MDR) and FEPS (MDR) cell lines. The K562 cells were more sensitive to UVA than the FEPS cells. The FEPS cell line was more resistant to H2O2 than K562, only presenting cytotoxicity 72 h after being exposed to 40 mM, with no ROS increase until 48 h. Both cell lines were sensitive to UVB, presenting cytotoxicity after 24 h, mainly by apoptosis, and showed an increase in ROS levels. Our results indicate that agents acting on DNA may be able to overcome the MDR phenotype.

Keywords: H2O2 , leukemia, MDR phenotype, UVA, UVB

Introduction

Chronic myeloid leukemia (CML) is a myeloproliferative clonal disease affecting hematopoietic stem cells undergoing the differentiation process within the bone marrow (Albano et al., 2013; Marcé et al., 2013). As in other cancers, the multiple drug resistance (MDR) phenotype can occur after chemotherapy treatment, with associated resistance to various drugs. These drugs may (or may not) be chemically related to distinct mechanisms of action operating in different cell targets (Gottesman and Pastan, 1993; Gottesman, 2002; Carret‐Dias et al., 2011). The MDR phenotype can present many characteristics, including an increase in membrane transporters such as ABCB1 (Pg‐p), ABCC1 (MRP), and ABCG2 (BCRP), DNA repair alterations, increased antioxidant capacity, cytoskeletal alterations, and microtubule rearrangements (Trindade et al., 1999; Gottesman, 2002; Votto et al., 2007; Daflon‐Yunes et al., 2013). Therefore, the MDR phenotype is non‐specific and multifactorial, resulting in the need for a better understanding of the mechanisms involved in tumoral resistance. In an attempt to better understand this phenotype, MDR cell lines were established from the parental K562 erythroleukemic cell line by treatment with two different chemotherapeutic drugs directed at two distinct cell targets. The MDR cell line K562‐Lucena 1 (Lucena) resulted from exposure to increasing concentrations of the chemotherapeutic drug vincristine (Rumjanek et al., 2001), which acts on microtubules. The MDR cell line FEPS was established by using increasing concentrations of the chemotherapeutic drug daunorubicin (Daflon‐Yunes et al., 2013), which directly induces DNA damage. Given these differences, it is highly relevant to investigate whether the cell target of the chemotherapeutic responsible for resistance induction will determine further responses to agents with different cell targets. Previous studies have shown that the MDR Lucena cell line was sensitive when challenged with agents aiming directly at the DNA as a target, whereas it was resistant to agents aimed at the membrane and the cytoskeleton and to oxidant agents (Trindade et al., 1999; Votto et al., 2007, 2010). With regard to agents that act on distinct targets, UVA radiation causes damage to the cell membrane by the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) (Black, 1987; de Gruijl, 2000). In contrast, UVB radiation mainly affects the DNA and can be directly absorbed by it (de Gruijl, 2000). Therefore, in the present work the sensitivity of the FEPS cell line to agents with different targets was evaluated. The aim of this study was to investigate whether there is any relationship between the cell target via which the cell resistance was induced and its sensitivity to agents with different cell targets.

Materials and methods

Cell culture

The K562 (non‐MDR) and FEPS cells (MDR) were obtained from the Tumoral Immunology Laboratory, placed at Leopoldo de Meis Medical Biochemistry Institute of the Federal University of Rio de Janeiro. The cell lines were maintained in RPMI 1640 (Gibco, USA) medium, supplemented with sodium bicarbonate (2 g/L) (Vetec, Brazil), l‐glutamine (0.3 g/L) (Vetec, Brazil), 10% of fetal bovine serum (Gibco, Brazil), 1% of antibiotics (100 U/mL of penicillin and 100 µg/mL de streptomycin) and antimycotic (0.25 µg/mL of amphotericin) (Gibco, USA). The cells were maintained in culture flasks at 37°C and 5% CO2 atmosphere. The FEPS cell line received 300 ng/mL of daunorubicin (DNR) (Sigma–Aldrich, Brazil) for preservation of the MDR phenotype (Daflon‐Yunes et al., 2013).

Exposure of erythroleukemic K562 and FEPS cell lines to UVA and UVB radiation

Cells (2.5 × 105 cells/mL) were exposed to different doses of UVA (0.2, 1, and 2 J/cm2) and UVB (0.01; 0.03; 0.06; and 0.12 J/cm2). To this, the culture medium was removed from the cells and replaced by PBS before placing the cells in Petri dishes (for UVA irradiation) and 24‐ or 96‐well plates (for UVB irradiation). Different exposure times were used to reach the desired doses. An UVA lamp (VL 115L: 115V, 30 W Vilber Lourmat, France) with an intensity peak of 365 nm and irradiance of 0.00375 J/cm2, and an UVB lamp (VL 115M: 115V, 30 W) with emission peak of 312 nm and irradiance of 59.09 μw/cm2 were used. After UVA irradiation, cells were transferred to 96‐well plates and further centrifuged at 5G during 8 min. After UVB irradiation, the 96‐well plates and 24‐well plates were centrifuged at 252 G for 5 min and 287 G for 10 min, respectively. The PBS was removed, the cells were resuspended in supplemented culture media and kept in an incubator at 37°C, with a 5% CO2 atmosphere, up to the necessary time for each reading or analysis.

Exposure of erythroleukemic K562 and FEPS cell lines to H2O2

Cells (2.5 × 105 cells/mL) were exposed to H2O2 (Alphatec, Brazil), at the concentrations of 10, 20, 30, and 40 mM for 30 min in culture media. After exposure, cells were rinsed in PBS and resuspended in culture medium without H2O2 and kept in an incubator at 37°C, with a 5% CO2 atmosphere, up to the necessary time for each experiment.

Cell viability

The cell viability was measured using trypan blue exclusion at time points 0, 24, 48, 72, and 96 h after exposure to the UVA, UVB, or H2O2. The percentage of cell viability was obtained by the number of viable cells divided by the total number of cells in a sample. Based on the results of these experiments, the dose 0.03 J/cm2 of UVB and the concentration of 30 mM of H2O2 were chosen for the other assays.

Quantitative analysis of cell death by apoptosis and necrosis

The evaluation of apoptosis and necrosis of the FEPS cell line was realized according Ribble et al. (2005) after 24 and 48 h following exposure to UVB or H2O2 in 96‐well plates. Acridine orange (100 µg/mL) and ethidium bromide (100 µg/mL) both from (Sigma, St. Louis, MO, USA) staining solutions were added to each well to be analyzed. The evaluation of cell death responses was realized based on photo‐documented areas from each well (400× magnification) using a fluorescence microscope (Olympus IX81). Data were expressed in relative percentage compared with the total number of cells visualized in the photo‐documented area. The cells were classified according to the modified method previously described of Kosmider et al. (2004). We consider viable the cells presenting green stained nucleus, while those evincing orange stained nucleus with fragmented chromatin and green cytoplasm were considered apoptotic. Cells with uniformly orange stained nucleus were considered necrotic.

Evaluation of reactive oxygen species (ROS)

The capability of UVB or H2O2 treatments to induce ROS production in FEPS cell line was evaluated after 24 h of exposure to these agents. The method used was generating ROS by heating the fluorescent compound 2′,7′‐dichlorofluorescin diacetate (H2DCF‐DA, Sigma, Germany) at a final concentration of 40 μM. The fluorescence was analyzed at 37°C for 1 h and 30 min. This experiment was analyzed in a fluorometer (Victor 2, Perkin Elmer), using excitation and emission wavelengths of 485 and 520 nm, respectively. ROS levels were expressed in terms of fluorescence area, after fitting fluorescence data to a second order polynomial and integrating between 0 and 90 min in order to obtain its area.

Data analysis

Three independent experiments were realized for all analyses and at least triplicates from the samples were used. All results were expressed as mean ± standard error, using one‐way ANOVA followed by Tukey's test and significance level of 0.05% (P < 0.05).

Results

Cell viability

The cell line K562 was shown to be sensitive to all UVA radiation doses immediately after exposure (0 h), exhibiting a reduction in cell viability (Figure 1A). However, no cytotoxicity was observed in the MDR FEPS cell line (Figure 1B).

Figure 1.

Figure 1

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Sensitivity to UVA radiation. Cell viability (%) of K562 (A) and FEPS (B) cell lines immediately (0), 24, 48, 72, and 96 h after UVA exposure (doses of 0.2, 1, and 2 J/cm2) by the trypan blue exclusion assay. Identical letters indicate absence of statistical differences (P < 0.05) at each time.

Concerning exposure to H2O2, the K562 cell line showed increased cytotoxicity after 24 h (Figure 2A) using the two higher concentrations (30 and 40 mM). On the other hand, the FEPS cell line showed a reduction in cell viability only at the concentration of 40 mM 72 h following exposure (Figure 2B).

Figure 2.

Figure 2

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Sensitivity to H2O2 Cell viability (%) of K562 (A) and FEPS (B) cell lines immediately (0), 24, 48, 72, and 96 h after exposure to H2O2 (concentrations of 10, 20, 30, and 40 mM) by the trypan blue exclusion assay. Identical letters indicate absence of statistical differences (P < 0.05) at each time.

When exposed to UVB, both cell lines were shown to be sensitive to all tested doses from 24 h after exposure, due to a reduction in cell viability (Figures 3A and 3B).

Figure 3.

Figure 3

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Sensitivity to UVB radiation. Cell viability (%) of K562 (A) and FEPS (B) cell lines immediately (0), 24, 48, 72, and 96 h after to UVB exposure (doses of 0.01, 0.03, 0.06, and 0.12 J/cm2) by the trypan blue exclusion assay. Identical letters indicate absence of statistical differences (P < 0.05) at each time.

Apoptosis and necrosis

No significant differences were observed in the percentage of cell death by apoptosis or necrosis, 24 and 48 h following exposure to H2O2, when compared with the untreated control group, in the MDR FEPS cell line. The same was true for the percentage of viable cells (Figure 4B).

Figure 4.

Figure 4

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Analysis of cell death. Percentage (%) of viable cells, apoptosis and necrosis after 24 and 48 h exposure to UVB radiation (0.03 J/cm2) (A), and after 24 and 48 h exposure to H2O2 (30 mM) (B). *, **, and *** show significant differences in the percentage of viable cells, apoptotic cells and necrotic cells, respectively. Results are expressed as mean ± standard error with P < 0.05.

A significant increase in cytotoxicity (P < 0.05), mostly by apoptosis, was observed in the MDR FEPS cell line 24 h after exposure to UVB radiation, although this cell line also presented a significant percentage of necrotic cells. The same pattern was seen after 48 h UVB exposure, when a significant difference (P < 0.05) was observed in the percentage of necrotic and apoptotic cells when compared to the control group. However, the number of necrotic cells was higher in the treatment group (Figure 4A).

ROS levels

No significant difference was found in the ROS level in the MDR FEPS cell line 24 h after exposure to 30 mM of H2O2 (Figure 5B). However, there was a significant increase in ROS concentration in the FEPS cell line after 24 h exposure to 0.03 J/cm2 of UVB (Figure 5A).

Figure 5.

Figure 5

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ROS levels (fluorescence area). Reactive oxygen species (ROS) generation in MDR FEPS cell line exposed to UVB radiation (0.03 J/cm2) (A) and to H2O2 (30 mM) (B) after 24 h. Asterisks indicate presence of significant differences (P < 0.05) in each group.

Discussion

The sensitivity of the MDR FEPS cell line to UVA and UVB radiation, as well as to H2O2, was evaluated based on the remaining viable cells, cell death, and reactive oxygen species (ROS) generation. The non‐MDR K562 cell line was also evaluated in order to compare parameters.

The K562 cell line was shown to be more sensitive to UVA radiation than MDR FEPS cells. Trindade et al. (1999) demonstrated a similar result in K562‐Lucena 1, reporting that the MDR cell line was more resistant to this radiation when compared with the parental K562 cell line, which was sensitive from the first day of exposure. The main products of UVA radiation are the ROS, such singlet oxygen (1O2) and hydrogen peroxide (H2O2) (de Gruijl, 2000; Ravanat et al., 2001). Trindade et al. (1999) tested the sensitivity of both cell lines to H2O2. Once again, the MDR K562‐Lucena1 cell line demonstrated higher levels of resistance than K562, with higher cell viability when challenged with high concentrations of H2O2 (from 10 to 80 mM). In the present work, the sensitivity to H2O2 was also tested in K562 and FEPS cells. And here again, the MDR cell line was more resistant than the non‐MDR cell line.

No significant difference was observed in the percentage of cell death by apoptosis or necrosis when the MDR FEPS cell line was exposed to H2O2. These data show that FEPS is more resistant to H2O2 than K562. The absence of cytotoxicity induced by H2O2 in FEPS is consistent with the work of Yamada et al. (1991). They reported that H2O2 resistant human leukemic cells (HP50‐2 and HP100‐1) presented higher expression of catalase, glutathione peroxidase (GPX), and superoxide dismutase (SOD) when compared with the non‐resistant cell line HL‐60.

Indeed, many authors have reported that MDR cells are more resistant to oxidative agents. While testing the toxin microcystin, which induces oxidative stress in cells, Votto et al. (2007) found that MDR K562‐Lucena 1 cells were more resistant than non‐MDR K562 cells. This finding is consistent with the results of Trindade et al. (1999), who found that UVA and H2O2, the agents responsible for inducing oxidative stress, had a greater effect on the non‐resistant K562 cell line than the resistant cell line. Both responses can be explained by the capacity of MDR cells to develop a more efficient antioxidant system than that presented in K562 cells. According Trindade et al. (1999) and Votto et al. (2007) the K562‐Lucena cells had high catalase activity. In fact there was no significant difference in the ROS level in the MDR FEPS cells after exposure to H2O2.

Acharya and Sahoo (2016) studied the relation between the redox imbalance and resistance to the chemotherapeutic drug Imatinib. They reported that K562R Imatinib‐resistant leukemic cells showed higher expression of the antioxidant enzymes SOD and catalase when compared with the K562 cell line. Therefore, the redox buffering of these enzymes can be considered the first defense mechanism of tumor cells subjected to stress induced by ROS (Acharya and Sahoo, 2016), supporting the hypothesis that resistant cell lines might present more efficient antioxidant strategies than non‐MDR cell lines.

Concerning UVB radiation, both the K562 and FEPS cell lines were shown to be sensitive, and the FEPS cells also showed a significant increase in the percentage of apoptotic and necrotic cells. The elevated apoptosis induced by UVB in FEPS was expected, since this irradiation wavelength (290–320 nm) induces mutagenic damage in the DNA molecule (Mouret et al., 2006). Thus, UVB is a death inductor agent for MDR FEPS cells.

Similar results were observed by Trindade et al. (1999) for the K562 and MDR K562‐Lucena 1 cell lines, with both being sensitive to a UVB dose of 0.03 J/cm2. Given that both MDR erythroleukemic cell lines (K562‐Lucena 1 and FEPS) were sensitive to UVB action, this agent might be able to transpose the MDR phenotype. Furthermore, the results obtained here reinforce the hypothesis that agents that are harmful to DNA integrity might overcome resistance in tumoral cells. The key to cancer treatment might be enclosed in this set of data, considering that agents usually induce apoptosis due to damage to the DNA, even in MDR cell lines. Indeed, some authors have speculated that agents directly targeting the DNA might be able to sensitize tumoral MDR cells similarly to non‐MDR (Trindade et al., 1999; Votto et al., 2010).

UVB radiation was capable of increasing the ROS level in FEPS cells. The ROS generated by UVB are, beyond singlet oxygen and H2O2 itself, hydroxyl radicals (Scharffetter‐Kochanek et al., 2000; Ravanat et al., 2001), which could explain the inability of the cell to avoid the oxidative stress in the same way that it fights against H2O2 only. Furthermore, UVB could have a combined effect from these ROS and the direct damage of the DNA, mainly via alterations in the binding of nucleotide bases, such as pyrimidine dimer formation (Ravanat et al., 2001). These alterations might lead the cells to an apoptotic cascade, which explains the higher cytotoxic induction and greater drop in FEPS cell proliferation when challenged by this agent.

Higher UVB efficiency also applies to K562, which although presenting cytotoxicity when challenged by all agents, was more sensitive to UVB. Therefore, agents that act on the DNA might cause higher cytotoxicity and induce an apoptotic cascade in a more effective manner than agents with other cell targets, regardless of whether the exposed cell lines are MDR or non‐MDR.

Conclusions

The results suggest that even when the target that induced the MDR phenotype in the FEPS cell line is the DNA, these cells remain sensitive to agents targeting this molecule. Thus, we conclude that agents acting directly on the DNA molecule, like UVB, could be more effective in overcoming the MDR.

Acknowledgments and funding

This work was funded by the Programa Nacional de Cooperação Acadêmica da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROCAD‐CAPES) [grant numbers 2951/2014]. FSS received a graduate fellowship from Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS).

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